All animal studies were conducted in accordance with the New South Wales Animals Act (1985). Approval was issued by The University of Sydney Animal Ethics Committee (Approval number: K17/9-2007/3/4664). All efforts were made to minimize animals suffering. The animals, Ins2Akita mice (Akita mice), were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The Akita mouse carries a dominant point mutation in the Insulin 2 gene on chromosome 7 resulting in the development of diabetes at approximately 4 weeks after birth with almost 100% penetrance. As female mice develop diabetes more slowly and less stably compared with males, only male mice heterozygous for the Ins2Akita allele (diabetic group) as well as male mice homozygous for the wild type Ins2 allele (non-diabetic mice) were used in this study. Presence of the Ins2Akita allele or the wild type Ins2 gene was confirmed by RFLP analysis. Once diabetes was established (blood glucose level > 13.3mmol/l), mice were monitored weekly for changes in body weight and blood glucose levels for 18 weeks. The blood glucose level was measured using Accu-Chek Performa (Roche, Germany). No supplemental insulin was given. Only mice having blood glucose levels consistently ≥ 13.3 mmol/l were used in this study. Eight diabetic and 8 non-diabetic mice were used.
After euthanizing mice with CO2, the femorae and tibiae of both legs were immediately excised and the diaphyses flushed using a 25g needle and 8 ml of IMag™ buffer (BD, Cat no. 552362). The collected cells were placed on ice. After centrifugation (400 rcf, 5 min), the cell pellet was resuspended in 2 ml red cell lysing buffer (Sigma, Cat no. R7757). After 5 min incubation and centrifugation (400 rcf, 5 min), the cell pellet was resuspended in 10 ml of IMag™ buffer and washed twice. Cells were eventually filtered using a 40 μm nylon cell strainer (BD, Cat no. 352340), centrifuged (400 rcf, 5 min) and resuspended in 2 ml of IMag™ buffer. After cell counting (TC10, BioRad) and viability assessment using the trypan blue exclusion assay, cells were placed on ice for the isolation of Lin-/VEGF-R2+ cells.
Immunomagnetic bead separation of BM Lin-/VEGF-R2+ endothelial progenitor cells
BM cells were incubated with NA/LE rat anti-mouse CD16/CD32 (Fc-block, 1 μg/106 cells, BD, Cat no. 553140) for 15 min. After Fc-block, BM cells were incubated with a solution containing an APC mouse lineage antibody cocktail (BD, Cat no. 558074) and a FITC rat anti-mouse Flk-1/VEGF-R2 antibody (BD, Cat no. 560680). After 20 min incubation, cells were centrifuged (400 rcf, 5 min) and washed twice using cold IMag™ buffer. Cells were incubated with magnetic beads for 30 min at 4°C (APC magnetic particles-DM; BD, Cat no. 557932). The APC mouse lineage cocktail was used to separate Lin+ cells from the whole of bone marrow cells, i.e. hematopoietic lineage cells such as T lymphocyes, B lymphocytes, monocytes/macrophages, granulocytes, and erythrocytes cells containing surface antigens such as CD3e, CD11, CD45R/B220, Ly-76, Ly-6G and Ly-6C. The Lin+ depletion was conducted using a Dynal MPC-S magnetic separator (Invitrogen, Cat no. 12020D). The Lin- fraction was further incubated with anti-FITC beads (Miltenyi Biotec, Cat no. 130-048-701) for 30 min at 4°C. The fraction of Lin-/VEGF-R2+ EPCs was obtained via a positive selection step using the magnetic separator. For RNA isolation, fractions of both Lin+ and the Lin-/VEGF-R2+ cells were collected, washed in PBS and centrifuged. Cell pellets were re-suspended in 100 μl of RNA later solution (Qiagen, Cat no. 76104) and snap-frozen in liquid nitrogen. Cells destined to undergo protein analysis were directly snap-frozen and stored at -80°C for further use.
Four experimental groups were established: 1) Lin+ cells from non-diabetic animals, 2) Lin+ cells from diabetic animals, 3) Lin-/VEGF-R2+ cells from non-diabetic animals and 4) Lin-/VEGF-R2+ cells from diabetic animals. The Lin+ cells were used as an internal reference to identify differential gene expression occurring not exclusively in Lin-/VEGF-R2+ cells. This setup allowed us to distinguish differential gene expression which specifically occurred in diabetic BM derived Lin-/VEGF-R2+ EPCs from that which may also occur in other phenotypes of BM cells. Hence, only significant changes in gene expression observed in diabetic vs. non-diabetic Lin-/VEGF-R2+ EPCs that did not occur in the Lin+ population were considered in the final analysis.
RNA isolation was performed at room temperature using the RNeasy Mini Kit (Qiagen, Cat no. 74104) according to the manufacturer’s instructions. Isolated RNA was snap-frozen and stored at -80°C for further use. RNA concentration was measured using a Nanodrop 1000 (Nanodrop Products, DE, USA), integrity was assessed using the BioRad Experion automated electrophoresis system (BioRad, CA, USA) on a RNA StdSens Chip (BioRad Cat no. 700–7159).
Reverse transcription was done using the iScript cDNA synthesis kit (BioRad, Cat no. 170–8890). In brief, 4 μl of 5× iScript reaction buffer, 1 μl iScript reverse transcriptase, 500 ng RNA and water up to a total reaction volume of 20 μl were mixed. The reverse transcription program was designed as follows: 25°C for 5 min, 42°C for 60 min, 85°C for 5 min followed by 4°C at a hold step. Reactions were performed in a PCR machine (HBPX220, Hybaid, UK). The finial 20 μl cDNA product was diluted into 160 μl total volume using MQ water.
Real time PCR
Primer sequences for RT-PCR were obtained fromhttp://pga.mgh.harvard.edu/primerbank and fromhttp://primerdepot.nci.nih.gov/. In silico analyses were performed to identify the amplicon size and suitability of the primer pairs. An overview of genes tested and primers used is shown in Additional file1: Table S1. All primers had a melting temperature of approximately 61°C and were tested before RT-PCR using gel electrophoresis to visualize amplicons. For testing primers, a total reaction volume of 10 μl comprised of 5 μl Super Mix (SsoFast EvaGreen Supermix, BioRad, Cat no. 172–5200), 1 μl of 4 μM forward and reverse primer mixture, 1 μl of cDNA and 3 μl water. PCR steps used were similar to the RT-PCR program used later: 95°C for 30 s, 40 cycles of 95°C for 5 s then 60°C for 20 s. This was followed by a melting curve step starting from 65°C to 95°C each step lasting 30s, ramp rate was 0.5°C/s. PCR products were analyzed in 2% agarose (in TBE buffer) gels to verify amplicon size.
RT-PCR was performed on a LightCycler 480 (Roche, Switzerland) using 384 well plates. Each group included seven individual samples, each individual sample was replicated once (technical replicate). The program was as follows: 95°C for 5 min, 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. Ramp rate was 4.8°C/s. Each well contained 5 μl Express Sybr Green (Invitrogen, Cat no. 10000162), 0.5 μl water, 0.5 μl of 4 μM forward and reverse primer mixture and 4 μl of the diluted sample cDNA. Mouse glyceraldehyde-3-phosphate dehydrogenase (mGAPDH)[32, 33] and 18S ribosomal RNA (18srRNA) were used as reference genes. The two reference genes were chosen using "BESTKEEPER" software (http://rest-2009.gene-quantification.info/), taking into account the information that there are no significant differences in mGAPDH and 18srRNA. Since progenitor cells from bone marrow were used, it was not clear whether one single chosen reference gene would be expressed. GAPDH content may be altered in animal models of diabetes, however, not all mouse strains are affected and in C57/BL6 mice mGAPDH has been successfully used as reference gene[32, 33]. CT-values were computed using the 2nd order derivation method, CT values ≥ 35 were excluded from the analysis. Data analysis was performed using the RT2 profiler PCR array data analysis available onhttp://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php.
Protein isolation and Western blot
For Western blot analysis, 8 samples from each group were used. The isolated cells were incubated and lysed for 30 min at 4°C in RIPA buffer (Sigma, Cat no. 127K6009) containing protease inhibitor (Complete mini; Roche, Cat no. 046931240010; 1 tablet per 10 ml RIPA buffer). Buffer volume was adjusted to a concentration of 5 × 104 cells/μl RIPA buffer. The lysed cells were centrifuged at 12,000 rcf for 20 min at 4°C. The supernatant containing the protein was aliquoted (26 μl) and stored at -20°C for further use.
Gel-electrophoresis to separate proteins according to their size was done using 2,2-Bis(hydroxymethyl)-2, 2′, 2″-nitrilotriethanol (Bis-Tris) polyacrylamide gels with a gradient from 4 to 12% under denaturing conditions (Nupage, Invitrogen, Cat no. NP0335) using 2-(N-morpholino) ethanesulfonic acid sodium dodecyl sulfate (MES-SDS, Invitrogen, Cat No NP0002) as running buffer. Before loading the gel wells, 26 μl protein sample, 10 μl loading buffer (Invitrogen, Cat no. NP0007), 4 μl 500 mM DL-dithiothreitol (DTT; Sigma, Cat no. D9779-10G) were mixed and kept at 70°C for 10 min to denature the protein and after this kept on ice for 5 min. After gel-electrophoresis, gels were removed from the running chamber and placed on a 0.2 μm polyvinyl difluoride (PVDF) membrane (Invitrogen, Cat no. LC2002). The protein transfer was done using a wet transfer system (BioRad Mini Trans-Blot, Cat no. 170–3930). After the transfer, the PVDF membranes were washed for 5 min using water and then for 10 min using TBST: tris (hydroxymethyl) aminomethane (TRIS) buffer, 150 mM sodium chloride and 0.1% polyoxyethylene (20) sorbitan monolaurate (TWEEN 20). A blocking step followed using 5% bovine serum albumin (BSA, Sigma, Cat no. 9418) in TBST and incubating the PVDF membrane for 1 h at room temperature. After washing the membrane twice in TBST, the incubation with the primary antibody (SDF-1; 1:2000, Abcam, Cat no. ab25117) in TBST and 1% BSA followed over night at 4°C. The next day the primary antibody solution was removed and the PVDF membrane washed 3 times in TBST for 5 min. Exposure to the secondary antibody HRP-goat anti-rabbit IgG (H + L) conjugate (horseradish peroxidase coupled; Zymed, Cat no. 81–6120) followed for 2 h at room temperature. After washing 3× for 5 min with TBST, the PVDF membrane was washed twice with TBS and then incubated for 5 min with the chemoluminescent agent (Millipore, Cat no. WBKLS0500). Immediately after this, the chemoluminescent agent was removed and the PVDF membrane was analyzed using a digital imaging system (G:Box, Syngene, MD, USA). After recording, the PVDF membrane was stripped of the antibodies by incubating for 5 min at room temperature with a Western blot stipping buffer (Thermo Scientific, Cat no. 46430). After washing the membrane in TBST, the incubation with the next primary antibody followed (E-Selectin, 1:2000, Abcam, Cat no. ab18981) and the procedure of overnight incubation, secondary antibody incubation and imaging was repeated. To evaluate differences in protein levels, chemiluminosity readings of target proteins were divided by chemiluminosity readings of reference proteins from the same sample. Relative expression levels were compared between diabetic and non-diabetic samples.
Data are presented as mean ± standard deviation (SD) for normally distributed data and as mean [interquartile range] when non-normally distributed. Normality was assessed using the Shapiro-Wilk test and the D’Agostino and Pearson Omnibus normality tests. Differences in variances of normally distributed data were assessed using Levene’s test. Differences between two groups were either assessed using a student’s t-test (normally distributed data) including Welch correction in case of unequal variances or Mann–Whitney test (non-normally distributed data). Statistical significance was defined as p < 0.05.