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. Author manuscript; available in PMC: 2023 Apr 5.
Published in final edited form as: Eur J Pharmacol. 2022 Feb 23;920:174842. doi: 10.1016/j.ejphar.2022.174842

Transforming growth factor-β1/Thrombospondin-1/CD47 axis mediates dysfunction in CD34+ cells derived from diabetic older adults

Jesmin Jahan 1, Ildamaris Monte de Oca 2, Brian Meissner 1, Shrinidh Joshi 1, Ahmad Maghrabi 2, Julio Quiroz-Olvera 2, Chrisitne Lopez-Yang 2, Stephen H Bartelmez 3, Charles Garcia 2, Yagna P Jarajapu 1
PMCID: PMC8967481  NIHMSID: NIHMS1788507  PMID: 35217004

Abstract

Aging with diabetes is associated with impaired vasoprotective functions and decreased nitric oxide (NO) generation in CD34+ cells. Transforming growth factor- β1 (TGF-β1) is known to regulate hematopoietic functions. This study tested the hypothesis that transforming growth factor- β1 (TGF-β1) is upregulated in diabetic CD34+ cells and impairs NO generation via thrombospondin-1 (TSP-1)/CD47/NO pathway. CD34+ cells from nondiabetic (ND) (n=58) or diabetic older adults (DB) (both type 1 and type 2) (n=62) were isolated from peripheral blood. TGF-β1 was silenced by using an antisense delivered as phosphorodiamidate morpholino oligomer (PMO-TGF-β1). Migration and proliferation in response to stromal-derived factor-1α (SDF-1α) were evaluated. NO generation and eNOS phosphorylation were determined by flow cytometry. CD34+ cells from older, but not younger, diabetics have higher expression of TGF-β1 compared to that observed in cells derived from healthy individuals (P<0.05, n=14). TSP-1 expression was higher (n=11) in DB compared to ND cells. TGFβ1-PMO decreased the secretion of TGF-β1, which was accompanied with decreased TSP-1 expression. Impaired proliferation, migration and NO generation in response to SDF-1α in DB cells were reversed by TGF-β1-PMO (n=6). TSP-1 inhibited migration and proliferation of nondiabetic CD34+ cells that was reversed by CD47-siRNA, which also restored these responses in diabetic CD34+ cells. TSP-1 opposed SDF-1α-induced eNOS phosphorylation at Ser1177 that was reversed by CD47-siRNA. These results infer that increased TGF-β1 expression in CD34+ cells induces dysfunction in CD34+ cells from diabetic older adults via TSP-1/CD47-dependent inhibition of NO generation.

Keywords: CD34+ cells, Transforming growth factor-β1, Thrombospondin-1, Nitric oxide, stromal-derived factor-1α

1. Introduction

CD34+ stem/progenitor cells have a strong therapeutic potential for the treatment of ischemic vascular complications. CD34+ cell therapy in patients with refractive angina significantly decreased the incidence of cardiac complications. (Johnson et al., 2020) However, this approach in individuals with long-term diabetes showed marginal success. (Jarajapu and Grant, 2010; Rigato et al., 2017) Diabetes impairs vasculogenic properties of CD34+ cells. Attenuated proliferation and migration to hypoxia-regulated factors such as stromal-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) are hallmark features of diabetic CD34+ cells. (Jarajapu et al., 2011) Nitric oxide (NO) generation in response to SDF-1α is decreased and increased generation of reactive oxygen species (ROS) was observed. (Jarajapu et al., 2011) Aging is an independent risk factor for reduced number and functional impairment of reparative cells, which correlate with vascular dysfunction and delayed or reduced recovery following ischemic insult. (Keymel et al., 2008; Umemura et al., 2008) Importantly, prevalence of diabetes is higher among older adults aged ≥65 years in US. (National Diabetes Statistics Report, 2020 | CDC) Prevalence of cardiovascular complications is three times higher in geriatric diabetic population compared to the nondiabetic. (Laiteerapong et al., 2019) Therefore, aging with diabetes has a greater impact on vasoreparative mechanisms compared to aging or diabetes alone.

Transforming growth factor-β1 (TGF-β1) is long-known for its pleiotropic functions in the embryonic and postnatal tissue homeostasis via cognate receptors type I and II receptors that are coupled with smad-dependent and -independent signaling pathways. (Guo and Wang, 2009; Nakagawa et al., 2004; Pardali et al., 2010) TGF-β1 signaling modulates HSC quiescence of hematopoietic stem/progenitor cells by preventing reentry of cells into the cell cycle. (Yamazaki et al., 2009) Aging stem/progenitor cells have increased expression of TGF-β1 receptors and myeloid bias, which contribute to aging-associated systemic inflammation and impaired vaso-regenerative potential. (Challen et al., 2010) Physiological levels of TGF-β1 in local or systemic environment are low, which could be elevated in pathologic conditions that would negatively impact hematopoietic and regenerative functions of CD34+ cells. (Chen et al., 2014; Ruscetti and Bartelmez, 2001) TGF-β1 receptors are associated with eNOS in caveolae and activation of receptors by TGF-β1 causes rapid dissociation and inactivation of eNOS. (Schwartz et al., 2005)

Thrombospondin-1 (TSP-1) is a matrix protein with anti-angiogenic propensity and positively regulated by TGF-β1. (Farberov and Meidan, 2018; Nakagawa et al., 2004) TSP-1 transduces signals by acting on cell surface receptors including but not limited to CD47, CD36 and integrins. (Lawler and Lawler, 2012; Resovi et al., 2014) Anti-angiogenic properties of TSP-1 were largely attributed to negative regulation of canonical NO signaling via activating CD47 receptor in a variety of tissues. (Isenberg et al., 2006a; Isenberg et al., 2006b)

Reversible silencing of TGF-β1 was shown to improve vascular repair-relevant functions in CD34+ cells derived from diabetic individuals such as increased proliferation and migration to SDF-1α that were associated with increased NO generation. (Bhatwadekar et al 2010) The current study tested the hypothesis that TGF-β1 expression is higher in CD34+ cells from diabetic adults, and that impaired NO generation is largely due to the inactivation of eNOS via TSP-1/CD47 pathway.

2. Methods

2.1. Characteristics of study subjects

The study was approved by the Institutional Review Board (IRB) and the Institutional Biosafety Committee (IBC) of North Dakota State University in accordance with the recommendations of the Helsinki declaration. The study was explained to all participants and all subjects have provided the consent to participate in the study. The following inclusion/exclusion criteria was used: Inclusion criteria: Age 21 years and above, both males and females, history of type 1 or type 2 diabetes, HbA1C level of 6.5 or greater, and any one of the following complications present: neuropathy, nephropathy, retinopathy, claudication/diabetic foot, myocardial ischemia or coronary artery disease. Exclusion criteria: Evidence of ongoing acute or chronic infection (HIV, Hepatitis B or C, tuberculosis, MRSA), ongoing malignancy, pregnancy or history of organ transplantation. The characteristics of study participants were listed in the Table 1. Whole blood samples were obtained by phlebotomy into vacutainer tubes (Museum District Eye Clinic, Houston, Texas). Nondiabetic leukocyte samples were obtained from a Leucoreduction chambers following apheresis by using the Trima Accel system (80440) (Vitalant, Fargo, North Dakota). The circulating CD34+ cells from male or female nondiabetic (n=58) or diabetic (n=62), both type 1 and type 2, individuals were used in the study.

Table 1:

Characteristics of study subjects

Nondiabetic (58) Diabetic (62)
Male/ Female 32/26 (33/29)
Age (years) 62±2 (26–85) 64±2 (38–86) NS
Ethnicity Caucasian/Hispanic Caucasian/Hispanic
Blood Glucose (mg/dL) 101±5 (97–149) 201±20 (180–548) (P<0.0001)*
HbA1C (%) 4.8±0.2 (4.9–6.1) 7.9±0.2 (6.5–14.0) (P<0.0001)*
Type 1 diabetes None 5
Type 2 diabetes None 57
Complications None Retinopathy (19), Neuropathy (12), Nephropathy (9), Hypertension (8), Erectile dysfunction (15) and Myocardial ischemia (5)
Medications - Metformin (41), Insulin (12), Statins (21), clopidogrel (11), Calcium channel blockers (14), β-blockers (8), gabapentin/pregabalin (12).
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Shown in parenthesis were the number of individuals; NS – Not significant

*

compared to nondiabetic

2.2. Isolation of CD34+ cells

Peripheral blood mononuclear cells (MNCs) were separated from total leucocytes by gradient centrifugation (800 g, 30 min) using Ficoll (Ficoll-Paque; GE Healthcare Biosciences) as described before. (Joshi and Jarajapu, 2019b) Buffy coats of MNCs were washed several times using PBS with 2% FBS and 1 mM EDTA and centrifuged at 120 g. MNCs were enriched for CD34+ cells using an immunomagnetic selection kit (Easysep, StemCell Technologies) as per the manufacturer’s instructions. The purity of enriched CD34+ cells was assessed by flow cytometry (C6 Accuri, BD) by using anti-CD45 and anti-CD34 antibodies (Biolegend) or isotype control antibodies in the presence of FcR blocking reagent (Milteny Biotech). (Joshi and Jarajapu, 2019b)

Freshly isolated CD34+ cells were either plated in U-bottom 96-well plates in StemSpan SFEM (StemCell Technologies) and used for functional studies on the following day. Where applicable cell pellets were preserved at −80°C for gene expression analysis. Silencing of TGF-β1 was accomplished by using Phosphorodiamidate Morpholino Oligomer (PMO). Control- or TGF-β1-PMO was purchased from Gene Tools (Philomath, OR, USA). Lyophilized PMOs were dissolved in sterile distilled water. CD34+ cells were incubated at 40 μg/mL PMO in StemSpan for 16–18 hours at 37°C and then excess PMO was removed by centrifugation with PBS. Then, the cells were either used for functional studies or flow cytometry or preserved at −80°C. Cells derived from an individual were treated with either Control- or TGF-β1 PMO in all assays for determining the impact of TGF-β1-silencing in comparison with that of control-treatment. Cells obtained from different subjects were randomly assigned to for different functional assays. Cells from both males and females were included in every assay. Where applicable, conditioned medium was collected, snap-frozen in liquid nitrogen and preserved at −80°C. Conditioned media were used for biochemical analysis of TGF-β1 ligand by ELISA (RnD Systems). For PMO influx and subcellular localization studies CD45dimCD34+ cells were obtained by fluorescence-activated cell sorting (FACS Aria, BD Biosciences). Lin− cells were clearly resolved into CD34+ and CD45+ populations using conjugated mouse anti-human antibodies, pacific blue–CD34 (BioLegend) and PE-Cy7-CD45 (BD Pharmingen).

2.3. Subcellular localization of fluoresceinated TGFβ1-PMO in CD34+ cells

CD34+ cells that were isolated and plated as described above, were treated with either 5 μg/ml or 40 μg/ml PMO with or without Endo-Porter (6 ug/ml) (Gene Tools, LLC, Philomath OR). After four or 20 hours, cells were pelleted at 400g for 5 min at 20 °C, the supernatant was discarded, and the cells were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4 for 10 min at 20 °C. After three washes in PBS, pelleting between washes, they were labeled with 1:500 Cy3 conjugated Goat-anti-Mouse IgG (Bethyl Laboratories) for 2 hours at 20 °C. Because the cells were isolated using anti-CD34 IgG conjugated to magnetic beads, their plasma membranes were labeled. Multi-well slides (Erie Scientific) were coated with poly-D-Lysine (Sigma) at 4°C overnight. The cells were washed again, the pellets were scraped with a pipette tip, and the cells were deposited onto the coated slides. The preparations were mounted in Vectashield anti-fade mounting medium with 4’,6-diamidino-2-phenylindole (DAPI, Vector Labs). Images of the labeled cells were obtained using a Zeiss LSM 780 confocal microscope. Illustrations were prepared using ZEN 2.1 (blue edition, Zeiss) and Photoshop® CS6 (Adobe). Cells were imaged in their entirety, a single optical section was selected, a Median filter was applied, and levels were adjusted.

2.4. Semi-quantitative real-time polymerase chain reaction (RT-PCR)

RNA was extracted from CD34+ cells using the Trizol method. The concentration and purity of RNA were determined by a spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). According to the manufacturer’s protocol, 1μg of RNA was reverse transcribed using a qScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). For detection of DNA synthesis in real-time, SYBR green was used. Each sample contained 50ng DNA, 10 μM of forward and reverse primers, and the iQ SYBR green containing supermix (Bio-Rad, Hercules, CA, USA). The specific primers for TGF-β1, TSP-1, and CD47 were synthesized by Invitrogen. A list of primer sequences is prosented in Table 2. β-actin was used as an internal housekeeping gene. The reactions were run in the Quantitative PCR System (Stratagene Mx3000P Multiplex Quantitative PCR System) using the following conditions: 3 min at 95°C, followed by 40 cycles of 10 sec at 95°C (denaturation step), 30 sec at 55°C (annealing step), and 30 sec at 72°C (extension step).

Table 2:

List of primers used for real-time PCR studies.

Primer Sequence
Gene Forward Reverse
TGF-β1 5’-GGACACCAACTATTGCTTCAG-3’ 5’-TCCAGGCTCCAAATGTAGG-3’
TSP1 5’-CACCAACCGCATTCCAGAG-3’ 5’-TCAGGGATGCCAGAAGGAG-3’
CD47 5’- AGATGTGGCCCTTGGGGC-3’ 5’-TGCTCAGACAACTGTATTC-3’
β-actin 5’-GACAGGATGCAGAAGGAGATTACT-3’ 5’-TGATCCACATCTGCTGGAAGGT-3’
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2.5. Transfection with siRNA

CD47 siRNA was used to knockdown CD47 expression in CD34+ cells, (Santa Cruz Biotech, catalog: sc-35006) and cells treated with a control siRNA (Santa Cruz Biotech, catalog: SC-37007) acted as a control group. Briefly, 2×105 CD34+ cells were transfected with 80 pmol of CD47siRNA or control siRNA with the transfection reagent (Santa Cruz Biotech, catalog: sc-29528) as per the manufacturer’s protocol. After 16 to 18 hours of incubation, media was replaced with StemSpan and plated for 48 or 72 hours. The efficiency of CD47 mRNA knockdown of cells after transfection was determined by qRT-PCR analysis with β-actin as a housekeeping gene.

2.6. Migration assay

The migration of CD34+ cells was evaluated by the QCM-Chemotaxis cell migration assay kit (EMD Millipore, Burlington, MA, USA) as per the manufacturer’s instructions. Briefly, 2×104 cells both untreated and CD47 siRNA treated were plated in the cell-migration chamber plate in the serum-free basal medium (HBSS) in the presence or absence of SDF-1α (100 nM), TSP-1 (30nM) in duplicate, and cells were allowed to migrate in response to the treatments for 5 h. After five hours, cells were dislodged from the membrane using cell detachment buffers, lysed, and quantified using fluorescent dye at 480/520 nm using a Spectramax plate reader (Molecular Devices). The migratory response was measured in arbitrary fluorescence units (Ex/Em: 480/525 nm) and expressed relative to the untreated.

2.7. Proliferation assay

The proliferation of CD34+ cells was evaluated using a kit that determined BrdU incorporation (Cell Proliferation ELISA; Roche Bioscience) as per the manufacturer’s instructions. The assay was performed by using 10 000 cells per treatment group, and each sample was tested in duplicate in basal medium StemSpan (Stemcell Technologies) and the proliferation was evaluated after 48 hours as per the manufacturer’s instructions. Absorbance was quantified at 350nm by using a Spectramax plate reader (Molecular devices). The results were expressed as fold increase in the absorbance relative to that observed with mitomycin (1 μM), which inhibits the proliferation.

2.8. Evaluation of NO generation and eNOS phosphorylation by flow cytometry

NO generation in CD34+ cells by SDF was evaluated by using DAF-FM, an NO-sensitive fluorescent probe and by quantifying the fluorescence by flow cytometer. (Joshi and Jarajapu, 2019a) Activation of eNSO by SDF was determined by flow cytometry of total eNOS, and phosphorylated eNOS, eNOS-Ser1177, and eNOS-Thr495, in CD34+ cells. (Joshi and Jarajapu, 2019a) First, cells were treated with FcR blocking reagent (Milteny Biotech) and then fixed and permeabilized with BD Cyto fix/Perm Reagent (BD Biosciences). Cells were then stained with antibodies, anti-eNOS (BD Bioscience, catalog: 610296), mouse anti-eNOS (pS1177) (BD Bioscience, catalog: 612393), and mouse anti-eNOS (pT495) (BD Bioscience, catalog: 612707) that were fluorescent-conjugated by Zenon-Alexa Fluor® 488 mouse IgG labeling kit, Zenon-Alexa Fluor® 647 mouse IgG labeling kit Zenon-Alexa Fluor® 700 mouse IgG labeling kit (Life Technologies), respectively, as per the manufacturer’s protocol. Cells were incubated for 45 min at 4°C and then washed with PBS before analyzed by flow cytometer. Both NO generation and eNOS phosphorylation were expressed as mean fluorescence intensity (MFI).

2.9. Drugs

SDF-1α and TSP-1 were purchased from R&D systems (Minneapolis, MN, USA), and stock solutions were made in PBS with 0.1% BSA and PBS only, respectively. PMOs and Endo-Porter were obtained from Gene Tools, LLC (Philomath OR).

2.10. Statistics

Data are presented as mean values ± SEM, and n represents the number of samples used. Results were analyzed for statistical significance by using the software program GraphPad (GraphPad Prism 5). Statistical differences among different treatment groups were assessed by using either paired or unpaired ‘t’-test or one-way ANOVA followed by Tukey’s post-test or two-way ANOVA followed by Bonferroni’s post-test for multiple comparisons, as applicable. Treatment groups were considered significantly different at P<0.05.

3. Results

3.1. Expression and secretion of TGF-β1 is higher in diabetic CD34+ cells

RT-PCR detected mRNA transcripts of TGF-β1 in both nondiabetic or diabetic CD34+ cells. The expression of TGF-β1 was higher in diabetic (n=12, P<0.05) compared to nondiabetic CD34+ cells (n=9) (Figure 1A) in individuals of age 60 years or older. No difference was observed at the age of 55 years or younger (n=7 for nondiabetic and diabetic) (Figure 1A). Therefore, rest of the study was carried out in cells derived from individuals of age 60 years or older. Secretion of TGF-β1 by diabetic cells (P<0.03) in older diabetic cells was higher compared to that observed in cells derived from age-matched nondiabetic individuals (n=8) (Figure 1B). Interestingly, ligand secretion was not observed in cells derived from five of ten nondiabetic individuals.

Figure 1. Gene expression and protein secretion of TGF-β1 were higher in CD34+ cells derived from diabetic older adults:

Figure 1.

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A. Expression of TGF-β1 mRNA, expressed as 2−ΔΔCt values relative to the expression of β-actin was higher in cells derived from older diabetics (DB) (P<0.01, n=12) compared to that in cells from the age matched nondiabetic (ND) cells (n=9). Expression was however similar in Young ND and DB groups. B. The secretion of TGF-β1 was higher in older DB cells (P<0.03) compared to the age-matched ND cells. Statistical analysis was carried out by unpaired ‘t’-test.

3.2. Influx and subcellular localization of PMO, and silencing of TGF-β1

FITC-TGF-β1-PMO was used to determine influx and within <75 minutes of incubation, 84±7% (n=10) (Figure 2A–2C) of CD34+ cells appeared positive for FITC as determined by flow cytometry. Secretion was determined in cells after incubating for 12 hours and conditioned medium was used to determine TGF-β1 concentration. Ligand secretion was decreased by 4- to 10-fold in cells that were treated with FITC-TGF-β1-PMO and the ligand was not detected in two of the five diabetic CD34+ cells after PMO-modification (n=5) (Figure 2D).

Figure 2. PMO trafficking in CD34+ cells:

Figure 2.

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A and B. Shown were representative flow cytometry dot plots showing CD45lowCD34+ cells treated with FITC-TGF-β1-PMO at different concentrations and evaluated for influx at 75 minutes following incubation. C. Summary of PMO uptake by CD34+ cells in a bar graph showing percent of FITC-positive cells following incubation with 0 or 40 μg/mL (n=10). D. Secretion of TGF-β1 ligand was decreased by 4- to 10-fold in CD34+ cells derived from three of the subjects while ligand was not detected in the other two subjects. E. Distribution of FITC-TGF-β1-PMO (green) in isolated CD34+ cells. The nuclei were labeled with DAPI (blue). PMO was applied for either 4 hours (left) or 20 hours (right). PMO concentrations were either 5 μg/mL or 40 μg/mL. In half of the conditions, Endo-Porter (6 μl/mL) was mixed with the PMO before application. In all conditions, there was punctate labeling throughout the cell. When the dose of PMO was 40 μg/mL, there were larger, round structures (arrows), and these were particularly prominent after 20 hrs.

Subcellular localization of PMO was evaluated by using fluoresceinated TGFβ1-PMO in CD34+ cells with or without treatment with Endo-Porter, a reagent designed to deliver PMO from acidic subcellular compartments into the cytosol (Figure 2E). FITC-TGFβ1-PMO was located in both the cytoplasm and the nucleus of the CD34+ cells under all conditions tested in PMO concentration-dependent fashion. At a dose of 5 μg/ml, very small punctuate areas, below the resolution limit of the light microscope, were labeled (Figure 2E). With a dose of 40 μg/ml, the pattern of small punctate areas plus larger, round structures, that became particularly prominent after 20 hours (Figure 2E). These subcellular compartments were very similar in untreated cells and cells treated with Endo-Porter. These findings suggest even at lower concentrations a relatively brief exposure is enough for uniform distribution of the PMO. Rest of the studies were carried out by using 40 μg/ml.

3.3. Silencing of TGF-β1 restores migration, proliferation and NO generation in response to SDF-1α in CD34+ cells derived from diabetic older adults

Migration was impaired in basal as well as in response to SDF-1α in CD34+ cells derived from diabetic older adults compared to the age-matched nondiabetics (P<0.05 and P<0.01, respectively) (Figure 3A). Control-PMO did not show any beneficial effect in diabetic cells. Pre-treatment with TGF-β1-PMO reversed dysfunction and restored migratory response to SDF-1α (100nM) (Figure 3A). Similar beneficial effects were observed in proliferation and NO generation in CD34+ cells following pre-treatment with TGF-β1-PMO compared with control-PMO. Cell proliferation and NO generation were lower in diabetic CD34+ cells in basal or in response to SDF-1α compared to that observed in the nondiabetic cells (P<0.05, n=6) ((Figures 3B and 3C)). Treatment with control-PMO did not show any improvement in the proliferation or NO-generation. Diabetic cells pre-treated with TGF-β1-PMO showed increased responses compared to untreated diabetic cells (P<0.001, n=6) (Figures 3B and 3C).

Figure 3. Silencing of TGF-β1 improved SDF-1α induced cell migration, proliferation, and NO generation in CD34+ cells from older diabetics:

Figure 3.

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A. Migration of cells was expressed as a percent increase relative to the response observed in the respective untreated control cells. Basal cell migration or in response to SDF-1α was impaired in diabetic (DB) CD34+ cells (P<0.05, n=6), compared to the nondiabetic cells. B. Proliferation was expressed as a fold-increase as compared with mitomycin that ws impaired in DB cells compared to that in ND cells in basal or in response to SDF-1α (P<0.05, n=6). TGF-β1-PMO increased SDF-1α-induced response in both ND (P<0.001) and DB cells (P<0.001) compared to the respective untreated or the control-PMO treated cells (n=6). C. NO generation was expressed as mean fluorescence intensity per treatment group. NO generation was impaired in DB cells (P<0.05) compared to nondiabetic cells (n=6) in basal (P<0.05) or in response to SDF-1α (P<0.001). Following modification with TGF-β1-PMO, NO generation was restored in DB cells in response to SDF-1α (P<0.001) compared to the untreated or Control-PMO-treated DB cells. Data was analyzed by One-way ANOVA with Tukey post-test.

3.4. Increased TSP-1 expression in diabetic CD34+ cells was reversed by TGF-β1-silencing

We tested the expression of TSP-1 in CD34+ cells from nondiabetic and diabetic older adults. TSP-1 expression was found to be higher in cells derived from diabetic older adults (n=14) compared to nondiabetics (P<0.05, n=18) (Figure 4A). As hypothesized, TSP-1 expression was decreased to nondiabetic levels after ex vivo modification with TGF-β1-PMO in diabetic cells compared to the Control-PMO treated cells (P<0.05, n=14) (Figure 4A). In nondiabetic cells, TGF-β1-PMO did not have significant effect on TSP-1 expression compared to the control-PMO treated cells (Figure 4A). Then, we tested the expression of TSP-1 receptor, CD47, in diabetic cells (n=9). No significant change was observed compared to that in nondiabetic cells (n=10) (Figure 4B).

Figure 4. TSP-1 gene expression is higher in CD34+ cells from older diabetics:

Figure 4.

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A. Expression of TSP-1 mRNA, expressed as 2−ΔΔCt values relative to the expression of β-actin, was higher in DB cells (P<0.05, n=18) compared to the ND cells (P<0.05, n=14). Modification with TGFβ-1-PMO decreased TSP-1 expression in DB cells to that observed in ND cells (P<0.05). No change was observed in ND cells following modification with TGFβ-1-PMO. B. The mRNA expression of CD47 was not different in cells derived from ND or DB subjects and was not changed by treatment with TGFβ-1-PMO (n=9). Statistical analysis was carried out by paired or unpaired ‘t’-test as applicable.

3.5. TSP-1 decreases proliferation in CD34+ cells via CD47 receptor

In CD34+ cells derived from nondiabetic individuals, TSP-1 (100 nM) inhibited SDF-1α induced cell proliferation (P<0.05) (Figure 5A) although basal proliferation was unaffected. Next, to confirm if this response was mediated by CD47 receptor, RNA-interference approach was used. CD47 knockdown with 60 pmol of siRNA was 18% and 56% (n=2) at 48 and 72 hours, respectively, in comparison with control siRNA treatment. Knockdown with 80 pmol of CD47 siRNA was 78 and 83% (n=2) at 48 and 72 hours, respectively. Therefore 80 pmol of CD47 siRNA was used for all functional assays. TSP-1-mediated decrease in proliferation was not observed in nondiabetic CD34+ cells with CD47-knockdown in basal or in response to SDF-1α (P<0.05, n=6) compared to that observed in control siRNA-treated cells (Figure 5B). Then, we tested if CD47-knockdown would restore proliferation in diabetic CD34+ cells. Proliferation in response to SDF-1α was not observed in diabetic cells, which was not significantly affected by treatment with TSP-1 (Figure 5C). However, CD47-knockdown resulted in increased proliferation in response to SDF-1α (P<0.05) (Figure 5E) compared to control siRNA-treated cells in the presence or in the absence of TSP-1.

Figure 5. Knockdown of CD47 stimulates proliferation in CD34+ cells derived from older diabetics.

Figure 5.

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</P/> A. Proliferation of cells, expressed as fold increase relative to the mitomycin (1 μM)-treated cells was enhanced by SDF-1α in nondiabetic (ND) cells (P<0.01 vs untreated cells, n=6) with control (Ctrl)-siRNA. SDF-1α (100 nM) induced cell proliferation was inhibited by TSP-1 (30 nM) (P<0.05, n=6). TSP-1 decreased proliferation induced by SDF-1α (P<0.05 vs SDF-1α response without TSP-1). Cell proliferation was increased in the presence of TSP-1 in CD47 siRNA treated cells compared to control siRNA treated ND cells in basal or in response to SDF-1 in the presence or absence of TSP1 (P<0.05, n=6). B. SDF did not induce cell proliferation in DB cells. In CD47 siRNA-treated DB cells, SDF1-1-induced proliferation wither in the presence (P<0.001) or in the absence of TSP-1 (P<0.001) compared to the respective Ctrl-siRNA-treated groups. Data was analyzed by Two-way ANOVA with Bonferroni post-test.

3.6. TSP-1 decreases migration in CD34+ cells via CD47 receptor

TSP-1 did not affect basal migration in CD34+ cells derived from nondiabetic individuals however attenuated SDF-1α-induced migratory response (P<0.0001, n=5) (Figure 6A). In cells with CD47 knockdown, in the presence of TSP-1 basal migration was increased and SDF-1α-response was disinhibited (P<0.001, n=5) (Figure 6B). In diabetic CD34+ cells, TSP-1 decreased basal migration compared to the untreated, which did not achieve statistical significance (Figure 6C). As described above, migration to SDF-1α was not observed in diabetic CD34+ cells, which was restored by CD47-siRNA knockdown (P<0.001, n=5) (Figure 6C). The inhibitory effect of TSP-1 on basal as well as SDF-1α-induced migration were also reversed by CD47 knockdown (P<0.001, n=5) (Figure 6C).

Figure 6. CD47-silencing stimulates migration in CD34+ cells derived from older diabetics.

Figure 6.

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A. Migration of cells, expressed as fold increase relative to the basal response in the untreated group, was increased by SDF-1α in nondiabetic (ND) cells (P<0.001 vs untreated group, n=6) with control (Ctrl)-siRNA. TSP-1 (30 nM) decreased migration induced by SDF-1α (P<0.001 vs SDF-1α response without TSP-1 in the presence of Ctrl-siRNA). Migration was higher in CD47 siRNA treated ND cells compared to control siRNA treated group in basal or in response to SDF-1α in the presence or absence of TSP1 (P<0.001, n=6). B. SDF-1α did not induce migration in DB cells. In CD47 siRNA-treated DB cells, SDF-1α-induced proliferation either in the presence (P<0.001) or in the absence of TSP-1 (P<0.001) compared to the respective Ctrl-siRNA-treated groups. CD47-siRNA increased basal migration in the presence of TSP-1 compared to Ctrl-siRNA-treated group (P<0.001, n=6). Data was analyzed by Two-way ANOVA with Bonferroni post-test.

3.7. TSP-1 decreases eNOS activation in CD34+ cells via CD47 receptor

SDF-1α induced phosphorylation of eNOS at Ser1177 as detected by flow cytometry in nondiabetic CD34+ cells (P<0.05, vs untreated cells, n=5) (Figures 7A and 7B). TSP-1 had no effect on basal phospho-eNOS-Ser1177 however decreased SDF-1α-induced phosphorylation (Figures 7A and 7B). CD47-knockdown reversed the inhibitory effect of TSP-1 on eNOS phosphorylation (P<0.05, vs Control-siRNA treated cells, n=5) (Figure 7A and 7B). CD34+ cells have basal levels of eNOS phosphorylation at Thr495 however this was not significantly altered by treatment with SDF-1α or by treatment with TSP-1 with or without CD47-silencing (Figures 7A). Therefore, SDF-1α-induced eNOS activation and NO generation are largely dependent on phosphorylation at Ser1177. Similar trends in the recovery of eNOS-activation were observed in cells derived diabetic older adults but due to limited availability of cells from this group of subjects this set of experiments could not be accomplished in a statistically acceptable number of subjects.

Figure 7. TSP-1 attenuated eNOS-Ser1177 phosphorylation in ND cells that was reversed by CD47-silencing.

Figure 7.

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A. Representative flow-cytometry histograms of eNOS, phospho-eNOSSer1177 and phospho-eNOSThr495 in nondiabetic untreated and CD47 siRNA treated nondiabetic CD34+ cells with different treatment. B. In control-siRNA treated nondiabetic cells, SDF induced p-eNOSSer1177 phosphorylation relative to the total eNOS was increased (P<0.01, n=5) compared to their basal level and inhibited (P<0.002) in presence of TSP-1. However, nondiabetic CD47-siRNA treatment reversed the inhibitory effect of TSP-1 and increased of eNOSSer1177 phosphorylation with SDF-1α in presence of TSP-1 (P<0.001 vs Control-siRNA-TSP-1-SDF, n=5). Data was analyzed by Two-way ANOVA with Bonferroni post-test.

4. Discussion

This study reports several novel findings. CD34+ cells derived from older diabetic individuals have increased expression of TGF-β1, which was not observed in younger diabetics. TSP-1 expression is upregulated in CD34+ cells derived from older diabetics that was TGF-β1-dependent. In vitro exposure to TSP-1 recapitulated diabetic dysfunction in nondiabetic CD34+ cells by negatively impacting proliferation, migration and NO generation by SDF-1α. Lastly, TSP-1-induced dysfunctions in CD34+ cells are CD47-dependent and CD47 silencing restored vascular repair-relevant functions in response to SDF-1α in CD34+ cells derived from diabetic older adults. Here, we have chosen PMO-approach to accomplish transient-silencing of TGF-β1, which is sufficient to increase proliferation of cells that is impaired in diabetic cells. Therefore, this study further confirms translational potential of this approach for enhancing revascularization outcomes of autologous cell therapies for vascular complications in diabetic older adults. PMO approach only delivers the antisense to the extranuclear compartment but does not involve in vivo administration. PMO-conjugation results in a unique way of rapid delivery as well as rapid efflux and transient nature of silencing is free from drawbacks that are associated with viral approaches. (Blank and Karlsson, 2015; Milone and O’Doherty, 2018; Sitnicka et al., 2016; Tsai et al., 2000)

TGF-β1 is known for its pro-inflammatory and fibrogenic actions in diabetes and other pathological conditions. (Qiao et al., 2017) Transcriptional targets of this cytokine in CD34+ cells were found to be dysregulated in bone marrow failure syndrome that could be reversed by pharmacological inhibitors, AVID200 and SD208, of TGF-β1 signaling. (Joyce et al., 2019) Aging and diabetes may be independently associated with increased expression of TGF-β1-R1 therefore the cells are sensitive to the signaling mechanisms that induce functional impairments including but not limited to decreased NO levels, which is known to mediate several vascular repair-relevant functions in vasculogenic cells. TGF-β1 was known to regulate pro- and anti-angiogenic growth factors in a variety of cell types. Down-regulation of SDF-1α expression in bone marrow stromal cells and that of CXCR4 expression in CD34+ cells by TGF- β1 have tremendous negative impact on the interaction and migration of CD34+ cells in the bone marrow. (Wright et al., 2003) In another study, TGF-β1 showed dose-dependent effects on SDF-1α-induced migration and adhesion, potentiation at lower and inhibition at higher concentrations. (Basu and Broxmeyer, 2005) Proliferation and migration in response to hypoxia-regulated factors is a major in vitro functional signature of vasoregenerative potential of CD34+ cells that were restored in dysfunctional diabetic cells by transient silencing of TGF-β1. Both of these functions were known to be largely regulated by NO signaling.

TSP-1 activates TGF-β1 from its latent inactive form thus enabling signaling via TGF-β1 receptors, which in turn induces expression of TSP-1 thus TGF-β1 activation and signaling are sustained in autocrine and paracrine fashion. TSP-1 is a matricellular protein with multiple domains in the sequence that can interact with multiple cellular receptors including CD36, proteoglycans, integrins and CD47. (Chen et al., 2000) With respect to vascular pathology, TSP-1/CD47 signaling inhibits cell cycle progression and induces cell senescence and death in endothelial cells. (Gao et al., 2016) Therefore, cellular effects and signaling mechanism triggered by TSP-1 considerably vary from one cell type to the other. CD47 is an important receptor and is a component of β3 integrin complex, expressed in blood cells and vascular cells.(Grimbert et al., 2006; Isenberg et al., 2009b) TSP-1/CD47 axis was first shown to produce antiangiogenic effects by limiting biological effects of NO via multiple mechanisms including inhibition of eNOS activation and opposition of NO-dependent activation of guanylyl cyclase and generation of cGMP.(Bauer et al., 2010; Miller et al., 2010) TSP-1 inhibited growth factor-dependent angiogenesis by inhibiting eNOS activation in microvascular endothelial cells. (Isenberg et al., 2009a) Consistent with these reports, TSP-1- or CD47-deficient mice showed increased NO/cGMP signaling and accelerated recovery of regional blood flow following ischemic insult. (Isenberg et al., 2007; Isenberg et al., 2009b) Angiocrine dysfunction observed in diabetic CD34+ cells (Jarajapu et al., 2014; Joshi et al., 2021; Stepanovic et al., 2003) Current study shows that upregulated autocrine TSP-1/CD47 signaling in diabetic CD34+ cells attenuate vascular repair-relevant functions via inhibiting NO generation thus establishing a role of TSP-1 in autocrine as well as paracrine dysfunction in diabetes. NO-independent effects of TGF-β1/TSP-1 on angiogenesis were also reported. TGF-β1 inhibits angiogenesis via upregulation of TSP-1 and SERPINE1. SERPINE1 encodes PAI-1, which negatively impact integrin-dependent cell survival, adhesion, proliferation and migration. (Mukai et al., 2007; Stefansson and Lawrence, 1996) Inhibition of PAI-1 resulted in increased neovascularization by stem/progenitor cells. (Xiang et al., 2004)

This study has some limitations as different assays were carried out in cells derived from different individuals, correlation analysis between two parameters such as TGFβ1 secretion vs NO generation could not be done. It would be difficult to obtain enough number of cells for the analysis of paracrine factors and carry out NO generation by flow cytometry of DAF-FM from a limited volume of blood sample that could be obtained from diabetic older adults. Along similar lines, flow cytometry of eNOS activation to SDF to determine the impact of TSP1 with or without CD47-silencing is required to be carried out in cells from the same individual which could not be accomplished in diabetic older adults but was carried out in cells from age-matched nondiabetic subjects.

Collectively, these findings suggest that ex vivo modification of TGF-β1 expression is sufficient to restore vascular repair-relevant functions in the dysfunctional CD34+ cells. Importantly, this study provided novel mechanistic basis involving TSP-1 and CD47 in the diabetic impairment of NO generation and the complete reversal of this by TGF-β1-silencing. As the ex vivo PMO-modification is an extremely a safer approach this concept can be rapidly transitioned towards clinical translation for enhancing vascular regenerative outcomes of autologous cell therapies for vascular complications in diabetic older adults. Autologous cell-based therapy is a best approach for the treatment of diabetic vascular complications however the success of this approach is limited due to inherent functional defects in the cells due to long-term diabetes. (Fadini et al., 2017) Ex vivo molecular approaches are needed to reverse diabetic dysfunction and enhance reparative outcomes of cell-based therapies. Transient silencing of TGF-β1 with PMO-aided delivery of antisense oligomers is a promising approach as it is safe and effective. Unlike small interference RNAs (siRNAs), binding of PMOs with nucleic acids does not lead to degradation but accomplish effective steric blockade of gene functions. (Summerton, 1999), which prevents possible adverse effects of TGF-β1-deficiency. (Blank and Karlsson, 2015) The study by providing mechanistic basis for the reversal of diabetic dysfunction by a simple and reversible molecular manipulation, strongly supports clinical translation of this approach for diabetic vascular diseases.

Acknowledgements

This work was partly supported by the National Institute of Aging (NIA) [AG056881]; and The Core Biology Facility at North Dakota State University was made possible by NIGMS [P30-GM 103332-01].

List of abbreviations

CXCR4

C-X-C chemokine receptor type 4

DAF-FM

4-amino-5-methylamino-2′,7′-difluorescein

eNOS

Endothelial nitric oxide synthase

FITC

Fluorescein isothiocyanate

HbA1C

Hemoglobin A1C

HBSS

Hanks′ Balanced Salt solution

HSPCs

Hematopoietic stem/progenitor cells

HUVECs

Human umbilical vein endothelial cells

MNCs

Mononuclear cells

NO

Nitric oxide

PAI-1

Plasminogen activator inhibitor-1

PMO

Phosphorodiamidate morpholino oligomer

SDF-1α

Stromal-derived factor-1α

siRNA

Small interfering RNA

TGF-β1

Transforming growth factor-β1

TSP-1

Thrombospondin-1

ROS

Reactive oxygen species

VEGF

Vascular endothelial growth factor

Footnotes

Declaration of Interests

Authors declare ‘no interests’.

Credit Authors Statement

YPRJ – Conception and design of work, interpretation of results and a major contributor in writing the manuscript.

SHB – Technical and conceptual consultations and review of manuscript.

JJ, BM, SJ - Data acquisition, analysis and drafting manuscript.

CG, IMdO, JQO, CLY and AM – Identification of and communication with eligible participants, obtaining consent, collection of samples, and record keeping.

All authors read and approved the final manuscript.

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References

  1. Basu S, Broxmeyer HE, 2005. Transforming growth factor-β1 modulates responses of CD34+ cord blood cells to stromal cell-derived factor-1/CXCL12. Blood 106, 485–493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauer EM, Qin Y, Miller TW, Bandle RW, Csanyi G, Pagano PJ, Bauer PM, Schnermann J, Roberts DD, Isenberg JS, 2010. Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial-dependent vasorelaxation. Cardiovascular research 88, 471–481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhatwadekar AD, Guerin EP, Jarajapu YP, Caballero S, Sheridan C, Kent D, Kennedy L, Lansang MC, Ruscetti FW, Pepine CJ, Higgins PJ, Bartelmez SH, Grant MB, 2010. Transient inhibition of transforming growth factor-beta1 in human diabetic CD34+ cells enhances vascular reparative functions. Diabetes 59, 2010–19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blank U, Karlsson S, 2015. TGF-beta signaling in the control of hematopoietic stem cells. Blood 125, 3542–3550. [DOI] [PubMed] [Google Scholar]
  5. Challen GA, Boles NC, Chambers SM, Goodell MA, 2010. Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-β1. Cell stem cell 6, 265–278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen C, Lei W, Chen W, Zhong J, Gao X, Li B, Wang H, Huang C, 2014. Serum TGF-β1 and SMAD3 levels are closely associated with coronary artery disease. BMC cardiovascular disorders 14, 1–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen H, Herndon ME, Lawler J, 2000. The cell biology of thrombospondin-1. Matrix Biology 19, 597–614. [DOI] [PubMed] [Google Scholar]
  8. Fadini GP, Ciciliot S, Albiero M, 2017. Concise Review: Perspectives and Clinical Implications of Bone Marrow and Circulating Stem Cell Defects in Diabetes. Stem Cells 35, 106–116. [DOI] [PubMed] [Google Scholar]
  9. Farberov S, Meidan R, 2018. Fibroblast growth factor-2 and transforming growth factor-beta1 oppositely regulate miR-221 that targets thrombospondin-1 in bovine luteal endothelial cells. Biology of reproduction 98, 366–375. [DOI] [PubMed] [Google Scholar]
  10. Gao F, Chiu S, Motan D, Zhang Z, Chen L, Ji H, Tse H, Fu Q-L, Lian Q, 2016. Mesenchymal stem cells and immunomodulation: current status and future prospects. Cell death & disease 7, e2062–e2062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grimbert P, Bouguermouh S, Baba N, Nakajima T, Allakhverdi Z, Braun D, Saito H, Rubio M, Delespesse G, Sarfati M, 2006. Thrombospondin/CD47 interaction: a pathway to generate regulatory T cells from human CD4+ CD25− T cells in response to inflammation. The Journal of Immunology 177, 3534–3541. [DOI] [PubMed] [Google Scholar]
  12. Guo X, Wang X-F, 2009. Signaling cross-talk between TGF-β/BMP and other pathways. Cell research 19, 71–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Isenberg JS, Hyodo F, Matsumoto K-I, Romeo MJ, Abu-Asab M, Tsokos M, Kuppusamy P, Wink DA, Krishna MC, Roberts DD, 2007. Thrombospondin-1 limits ischemic tissue survival by inhibiting nitric oxide–mediated vascular smooth muscle relaxation. Blood 109, 1945–1952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Isenberg JS, Martin-Manso G, Maxhimer JB, Roberts DD, 2009a. Regulation of nitric oxide signalling by thrombospondin 1: implications for anti-angiogenic therapies. Nature Reviews Cancer 9, 182–194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Isenberg JS, Qin Y, Maxhimer JB, Sipes JM, Despres D, Schnermann J, Frazier WA, Roberts DD, 2009b. Thrombospondin-1 and CD47 regulate blood pressure and cardiac responses to vasoactive stress. Matrix Biology 28, 110–119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Isenberg JS, Ridnour LA, Dimitry J, Frazier WA, Wink DA, Roberts DD, 2006a. CD47 is necessary for inhibition of nitric oxide-stimulated vascular cell responses by thrombospondin-1. Journal of Biological Chemistry 281, 26069–26080. [DOI] [PubMed] [Google Scholar]
  17. Isenberg JS, Wink DA, Roberts DD, 2006b. Thrombospondin-1 antagonizes nitric oxide-stimulated vascular smooth muscle cell responses. Cardiovascular research 71, 785–793. [DOI] [PubMed] [Google Scholar]
  18. Jarajapu YP, Caballero S, Verma A, Nakagawa T, Lo MC, Li Q, Grant MB, 2011. Blockade of NADPH oxidase restores vasoreparative function in diabetic CD34+ cells. Invest Ophthalmol Vis Sci 52, 5093–5104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jarajapu YP, Grant MB, 2010. The promise of cell-based therapies for diabetic complications: challenges and solutions. Circ Res 106, 854–869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jarajapu YP, Hazra S, Segal M, LiCalzi S, Jhadao C, Qian K, Mitter SK, Raizada MK, Boulton ME, Grant MB, 2014. Vasoreparative dysfunction of CD34+ cells in diabetic individuals involves hypoxic desensitization and impaired autocrine/paracrine mechanisms. PloS one 9, e93965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Johnson GL, Henry TD, Povsic TJ, Losordo DW, Garberich RF, Stanberry LI, Strauss CE, Traverse JH, 2020. CD34+ cell therapy significantly reduces adverse cardiac events, health care expenditures, and mortality in patients with refractory angina. Stem Cells Translational Medicine 9, 1147–1152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Joshi S, Jarajapu YP, 2019a. Mitochondrial depolarization stimulates vascular repair-relevant functions of CD34+ cells via reactive oxygen species-induced nitric oxide generation. British journal of pharmacology 176, 4373–4387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Joshi S, Jarajapu YPR, 2019b. Mitochondrial depolarization stimulates vascular repair-relevant functions of CD34(+) cells via reactive oxygen species-induced nitric oxide generation. Br J Pharmacol 176, 4373–4387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Joshi S, Montes de Oca I, Maghrabi A, Lopez-Yang C, Quiroz Olvera J, Garcia CA, Jarajapu YP, 2021. ACE2 Gene Transfer Ameliorates Vasoreparative Dysfunction in CD34+ Cells Derived from Diabetic Older Adults. Clinical Science. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Joyce CE, Saadatpour A, Ruiz-Gutierrez M, Bolukbasi OV, Jiang L, Thomas DD, Young S, Hofmann I, Sieff CA, Myers KC, 2019. TGF-β signaling underlies hematopoietic dysfunction and bone marrow failure in Shwachman-Diamond syndrome. The Journal of clinical investigation 129, 3821–3826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Keymel S, Kalka C, Rassaf T, Yeghiazarians Y, Kelm M, Heiss C, 2008. Impaired endothelial progenitor cell function predicts age-dependent carotid intimal thickening. Basic Res Cardiol 103, 582–586. [DOI] [PubMed] [Google Scholar]
  27. Laiteerapong N, Ham SA, Gao Y, Moffet HH, Liu JY, Huang ES, Karter AJ, 2019. The legacy effect in type 2 diabetes: impact of early glycemic control on future complications (the Diabetes & Aging Study). Diabetes Care 42, 416–426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lawler PR, Lawler J, 2012. Molecular basis for the regulation of angiogenesis by thrombospondin-1 and-2. Cold Spring Harbor perspectives in medicine 2, a006627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Miller TW, Isenberg JS, Shih HB, Wang Y, Roberts DD, 2010. Amyloid-β inhibits No-cGMP signaling in a CD36-and CD47-dependent manner. PloS one 5, e15686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Milone MC, O’Doherty U, 2018. Clinical use of lentiviral vectors. Leukemia 32, 1529–1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mukai Y, Wang C-Y, Rikitake Y, Liao JK, 2007. Phosphatidylinositol 3-kinase/protein kinase Akt negatively regulates plasminogen activator inhibitor type 1 expression in vascular endothelial cells. American Journal of Physiology-Heart and Circulatory Physiology 292, H1937–H1942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nakagawa T, Li JH, Garcia G, Mu W, Piek E, Böttinger EP, Chen Y, Zhu HJ, Kang D-H, Schreiner GF, 2004. TGF-β induces proangiogenic and antiangiogenic factorsvia parallel but distinct Smad pathways. Kidney international 66, 605–613. [DOI] [PubMed] [Google Scholar]
  33. Pardali E, Goumans M-J, ten Dijke P, 2010. Signaling by members of the TGF-β family in vascular morphogenesis and disease. Trends in cell biology 20, 556–567. [DOI] [PubMed] [Google Scholar]
  34. Qiao Y-C, Chen Y-L, Pan Y-H, Ling W, Tian F, Zhang X-X, Zhao H-L, 2017. Changes of transforming growth factor beta 1 in patients with type 2 diabetes and diabetic nephropathy: a PRISMA-compliant systematic review and meta-analysis. Medicine 96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Resovi A, Pinessi D, Chiorino G, Taraboletti G, 2014. Current understanding of the thrombospondin-1 interactome. Matrix Biology 37, 83–91. [DOI] [PubMed] [Google Scholar]
  36. Rigato M, Monami M, Fadini GP, 2017. Autologous Cell Therapy for Peripheral Arterial Disease: Systematic Review and Meta-Analysis of Randomized, Nonrandomized, and Noncontrolled Studies. Circ Res 120, 1326–1340. [DOI] [PubMed] [Google Scholar]
  37. Ruscetti FW, Bartelmez SH, 2001. Transforming growth factor β, pleiotropic regulator of hematopoietic stem cells: potential physiological and clinical relevance. International journal of hematology 74, 18–25. [DOI] [PubMed] [Google Scholar]
  38. Schwartz EA, Reaven E, Topper JN, Tsao PS, 2005. Transforming growth factor-β receptors localize to caveolae and regulate endothelial nitric oxide synthase in normal human endothelial cells. Biochemical Journal 390, 199–206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sitnicka E, Storey C, Bartelmez S, 2016. Functional Resolution of Long-Term-and Short-Term-Hematopoietic Stem Cells. J Stem Cell Res Ther 1, 00044. [Google Scholar]
  40. Stefansson S, Lawrence DA, 1996. The serpin PAI-1 inhibits cell migration by blocking integrin α v β 3 binding to vitronectin. Nature 383, 441–443. [DOI] [PubMed] [Google Scholar]
  41. Stepanovic V, Awad O, Jiao C, Dunnwald M, Schatteman GC, 2003. Leprdb diabetic mouse bone marrow cells inhibit skin wound vascularization but promote wound healing. Circulation research 92, 1247–1253. [DOI] [PubMed] [Google Scholar]
  42. Summerton J, 1999. Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1489, 141–158. [DOI] [PubMed] [Google Scholar]
  43. Tsai S, Fero J, Bartelmez S, 2000. Mouse Jagged2 is differentially expressed in hematopoietic progenitors and endothelial cells and promotes the survival and proliferation of hematopoietic progenitors by direct cell-to-cell contact. Blood, The Journal of the American Society of Hematology 96, 950–957. [PubMed] [Google Scholar]
  44. Umemura T, Soga J, Hidaka T, Takemoto H, Nakamura S, Jitsuiki D, Nishioka K, Goto C, Teragawa H, Yoshizumi M, Chayama K, Higashi Y, 2008. Aging and hypertension are independent risk factors for reduced number of circulating endothelial progenitor cells. Am J Hypertens 21, 1203–1209. [DOI] [PubMed] [Google Scholar]
  45. Wright N, de Lera T.L.n., García-Moruja C, Lillo R, García-Sanchez F.l., Caruz A, Teixidó J, 2003. Transforming growth factor-β1 down-regulates expression of chemokine stromal cell–derived factor-1: functional consequences in cell migration and adhesion. Blood 102, 1978–1984. [DOI] [PubMed] [Google Scholar]
  46. Xiang G, Schuster MD, Seki T, Kocher AA, Eshghi S, Boyle A, Itescu S, 2004. Down-regulation of plasminogen activator inhibitor 1 expression promotes myocardial neovascularization by bone marrow progenitors. The Journal of experimental medicine 200, 1657–1666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yamazaki S, Iwama A, Takayanagi S. i., Eto K, Ema H, Nakauchi H, 2009. TGF-β as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation. Blood 113, 1250–1256. [DOI] [PubMed] [Google Scholar]

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