Stem cell compartmentalization in diabetes and high cardiovascular risk reveals the role of DPP-4 in diabetic stem cell mobilopathy
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- Fadini, G.P., Albiero, M., Seeger, F. et al. Basic Res Cardiol (2013) 108: 313. doi:10.1007/s00395-012-0313-1
Bone marrow (BM) derived stem and progenitor cells contribute to cardiovascular homeostasis and are affected by cardiovascular risk factors. We devised a clinical data-driven approach to test candidate stem cell mobilizing mechanisms in pre-clinical models. We found that PB and BM CD34+ cell counts were directly correlated, and that most circulating CD34+ cells were viable, non-proliferating and derived from the BM. Thus, we analyzed PB and BM CD34+ cell levels as a two-compartment model in 72 patients with or without cardiovascular disease. Self-organizing maps showed that disturbed compartmentalization of CD34+ cells was associated with aging and cardiovascular risk factors especially diabetes. High activity of DPP-4, a regulator of the mobilizing chemokine SDF-1α, was associated with altered stem cell compartmentalization. For validation of these findings, we assessed the role of DPP-4 in the BM mobilization response of diabetic rats. Diabetes differentially affected DPP-4 activity in PB and BM and impaired stem/progenitor cell mobilization after ischemia or G-CSF administration. DPP-4 activity in the BM was required for the mobilizing effect of G-CSF, while in PB it blunted ischemia-induced mobilization. Indeed, DPP-4 deficiency restored ischemia (but not G-CSF)-induced stem cell mobilization and improved vascular recovery in diabetic animals. In conclusion, the analysis of stem cell compartmentalization in humans led us to discover mechanisms of BM unresponsiveness in diabetes determined by tissue-specific DPP-4 dysregulation.
Bone marrow (BM) derived stem and progenitor cells contribute to cardiovascular homeostasis through several mechanisms including differentiation into vascular phenotypes and paracrine signals [3, 22, 23, 30, 31]. The cross-talks between cardiovascular disease (CVD) and the BM system are manifold. Signals from ischemic tissues activate BM metabolism and function  and inform the BM for the need of vasculo-regenerative cells through soluble mediators (e.g., VEGF and SDF-1α) produced locally . These cross-talks are disturbed in the presence of cardiovascular risk factors , which are associated with reduced and dysfunctional BM-derived circulating progenitor cells, a mechanism that contribute to development and progression of CVD . This is particularly evident in diabetes, as progenitor cell alterations have been described extensively and consistently in animals and humans [12, 19]. BM defects in diabetes are being elucidated and include microangiopathy, neuropathy, altered gene expression and niche dysfunction, which hamper stem cell mobilization [7, 10, 21, 24, 25]. Yet, data on BM function in humans with diabetes or CVD are scant and difficult to collect due to the intrinsic low availability of BM samples.
In this study, we aimed to gather information on the clinical phenotype associated with an altered compartmentalization of stem/progenitor cells in the BM and peripheral blood (PB), which would be indicative of the mobilization capacity. Starting from these phenotypes, we devised a clinical data-driven approach to test the association of candidate regulators of angiogenesis with progenitor cell mobilization, to be validated in animal models.
Materials and methods
An expanded version of the method section can be found in the online supplemental data file.
Human studies were approved by the ethical committee of the Goethe University of Frankfurt and by the University Hospital of Padova and conducted in accordance to the Declaration of Helsinki, as revised in 2000. All subjects provided informed consent. Patients were recruited at the University Hospital of Frankfurt am Main. All patients undergoing BM aspiration for myocardial cell therapy in the trials NCT00962364 and NCT00284713 were eligible—pending they did not have acute myocardial infarction. Control subjects underwent BM and PB sampling, but did not receive cell therapy. We collected demographic, anthropometric data, cardiovascular risk factors, prevalence of CVD and medications.
Measurement of regulators of angiogenesis and DPP-4 activity
The plasma concentration of angiogenesis-regulating mediators was determined using multiplex suspension arrays (Bio-plex, Biorad). DPP-4 activity was measured with the DPP-4 drug discovery Kit (Enzo Life Sciences, Farmingdale, NY, USA).
Human circulating progenitor cells were quantified using flow cytometry by staining for CD34, CD133 and KDR. The proliferation rate of circulating CD34+ cells was determined by staining fixed and permeabilized cells with propidium iodide (PI) for DNA content estimation. The apoptotic rate of circulating CD34+ cells was analyzed by the Annexin-V binding assay and PI staining. The levels of rat stem and progenitor cells were quantified by flow cytometry by staining for Sca-1 and c-kit (hematopoietic stem/progenitor cells) or Sca-1 and CD31 (EPCs).
The animal protocol was approved by the ethic committee for animal experiments of the University of Padova and from the Italian Ministry of health. All experiments were conducted according to the “Principles of laboratory animal care” (NIH publication no. 85–23, revised 1985. We used 8-week-old F344 wild type and DPP-4 deficient rats. Diabetes was induced by intraperitoneal injection of streptozotocin 20 mg/kg. Experiments were performed 5–7 weeks after induction of diabetes. Hind limb ischemia was induced by femoral artery and vein excision. Blood samples were obtained before and on days 3 and 7 after ischemia. On day 7, rats were euthanized and for collection of muscles for histologic analysis of capillary density.
For direct bone marrow stimulation, rats were treated with human recombinant G-CSF at the dose of 60 μg/kg/day subcutaneously for four consecutive days.
Data are expressed as mean ± standard error or as percentage. Comparison between two or more groups was performed using Student’s t test or ANOVA (for normal variables) and with Mann–Whitney’s U test and Kruskal–Wallis test (for non-normal variables). Linear correlations were analyzed using the Pearson’s r (for normal variables) or the Spearman’s rho (for non-normal data) correlation coefficients.
Groups of patients were created according to their progenitor cell levels as below (termed “low”) or above (termed “high”) the median value in the peripheral blood and in the bone marrow. To render the separation of patients’ groups more extreme, the 50° percentile cutoff was substituted with the 60° percentile cutoff as lower limit of the “high” groups and with the 40° percentile as the higher limit of the “low” groups. The resulting four groups were compared using ANOVA or Kruskal–Wallis test and then with appropriate test comparing couples of groups.
The distribution of clinical characteristics in the four groups was also analyzed using self-organizing-maps (SOM) and statistically verified visualizations in the whole cohort and in patients belonging to the above mentioned groups defined by the median values or by the 40°/60° percentile. The SOM is an unsupervised pattern recognition method used for automated comparisons in a multivariate profile of clinical characteristics. In the map layout, patients with similar clinical characteristics are as close to each other, whereas those who have different characteristics are placed far apart on the map. After computing patients’ positions, the map is colored according to the patients’ characteristics. Statistical significance was accepted at p < 0.05 and SPSS ver. 16.0 was used.
Direct correlations between peripheral blood and bone marrow stem/progenitor cell levels
Most circulating CD34+ cells are viable, non-proliferating and originated from the bone marrow
As also circulating mature endothelial cells may express CD34 , we analyzed the origin of CD34+ cells isolated from three female patients who received a male BM transplantation years before, by detecting the Y-chromosome signal using FISH. After adjusting for hybridization efficiency (89 %) , we found that 92.5 ± 3.1 % CD34+ cells were Y+, indicative of a donor BM origin (Fig. 1b). Therefore, the contamination by non-BM-derived cells within the population of circulating CD34+ cells is <10 %. In samples of healthy blood donors, we also show that the apoptotic rate of circulating CD34+ cells is on average 4.0 ± 0.6 % and most are resting (>95 % in G0–G1 phase) (Fig. 1c). Therefore, variations in the number of PB CD34+ cells are mostly attributable to mobilization of BM CD34+ cells.
Distribution of PB and BM CD34+ cells
Stem cell compartmentalization and the cardiovascular risk profile
The four groups identified by the 40°/60° percentile of PB and BM CD34+ cell counts were first analyzed for demographics, cardiovascular risk factors and therapies using conventional statistics. We found that subjects in the “healthy” group were (not significantly) younger, had a low number of cardiovascular risk factors (p = 0.02), and a low prevalence of diabetes, obesity and hypertension compared with patients in the “exhausted” group (post-ANOVA tests). The other groups, in which the compartmentalization of CD34+ cells was altered, were associated with higher prevalence of cardiovascular risk factors, and the “exhausted” group showed the worst risk profile. Among therapies, use of beta-blockers and insulin were higher in the group of “good mobilizers”, consistent with the previous findings that these drugs can increase circulating progenitor cell levels [20, 27] (Fig. 2).
Compartmentalization was then repeated for other progenitor cell phenotypes (online Table 4S). Altered PB versus BM CD133+ and CD34+CD133+ cell compartmentalization was associated with a high cardiovascular risk profile similar to what obtained with CD34+ cell compartmentalization. However, the discriminative capacity of CD34+ cells was higher, as there were more clinical features differing among groups. These data confirm that CD34+ cell compartmentalization offers the best correlate of cardiovascular risk.
Stem cell compartmentalization and regulators of angiogenesis
We quantified the plasma concentrations of a panel of angiogenesis-regulating factors (online Table 4S). When patients were divided according to CD34+ cell compartmentalization, plasma VEGF concentrations and DPP-4 activity were lower in the “healthy” group, while other factors were not significantly different. SOM showed that high activity of the enzyme DPP-4 colocalizes with the “exhausted” phenotype (Fig. 2S).
The “healthy” group according to CD133+ and CD34+CD133+ cell compartmentalization had higher concentrations of the pro-angiogenic factors Angiopoietin-2 and PDGF-BB. Compartmentalization of CD34+CD133+KDR+ cells revealed high SDF-1α concentrations in the “healthy” and “good mobilizer” groups, consistent with the notion that circulating SDF-1α mobilizes EPCs . These data indicate that some mobilizing factors may explain progenitor cell compartmentalization in this cohort of patients. We also suggest that this clinical data-driven approach is a suitable platform to identify new strategies of progenitor cell mobilization.
We then aimed at validating one of these factors in diabetes, as a model of altered progenitor cell mobilization.
DPP-4 deficiency restores ischemia- but not G-CSF-induced mobilization in diabetes
This study, by combining a clinical data-driven discovery approach with animal models, shows that altered mobilization of stem/progenitor cells is associated with a high cardiovascular risk profile and, in the setting of diabetes, is driven by a tissue-specific DPP-4 dysregulation.
Progenitor cell mobilization, diabetes and cardiovascular risk
Investigation into the mechanisms of reduced progenitor cell levels has led to several hypothesis including excess apoptosis and senescence. In type 2 diabetes, we have previously shown that apoptosis is not responsible for the observed low level of circulating CD34+ cells . Rather, animal models suggest that BM alterations account for depressed circulating progenitor cells in diabetes [7, 21, 24]. Unfortunately, the poor availability of BM samples limits our understanding of BM function in human diabetes. Herein, we analyzed BM and PB samples from patients undergoing cell therapy for heart disease and healthy volunteers. The direct correlation between BM and PB progenitor cell levels, although scattered, indicates that circulating progenitor cell levels are informative of the BM status. We also show that most circulating CD34+ cells are BM-derived, non-apoptotic and non-proliferating. As long as homing to the target tissues is not increased , these data indicate that variations in circulating CD34+ cells among patients are attributable to different capacity of BM mobilization. This allowed to categorize patients according to their BM and PB progenitor cell status into distinct mobilizer phenotypes. This original approach, although based on a snapshot of a dynamic system, led to the discovery that individuals with an altered distribution of CD34+ cells in the BM and PB display a strikingly high cardiovascular risk profile, characterized by aging, diabetes, obesity and hypertension. Using self-organizing maps, we visually and statistically analyzed cardiovascular risk factors in relation to the mobilizer phenotype: DM was strongly associated with exhaustion of PB and BM CD34+ cells independently of the distribution of other risk factors. While reduction of circulating progenitors is a consistent finding in animal and human DM, the effect of DM on the amount of BM progenitors is debated. Long-term diabetic mice appear to have reduced BM Sca-1+c-kit+ hematopoietic stem cells [24, 25], while short-term diabetic rats showed no reduction of BM Sca-1+c-kit+  and short-term diabetic mice had normal  or even increased BM Sca-1+c-kit+ . While methodological issues can explain these discrepancies, we suggest that the low CD34+ cell count in BM aspirates of DM patients indicates a reduced accessibility of stem cell niches to the aspiration manoeuver, in compliance with the observation that the diabetic BM niche is sticky and more prone to stem cell retention than mobilization .
Among other clinical features, it appears that use of beta-blockers and insulin was associated with a good mobilizer phenotype. Experimental studies support a role for beta-blockers  and insulin therapy [11, 20] in improving progenitor cell mobilization, further indicating that the clinical phenotype associated with stem cell compartmentalization is biologically plausible.
Thanks to their instructing spatial networks, SOM provide complementary and incremental information over classic statistical group comparison, also indicating that the “good mobilizer” and, to a lesser extent, the “poor mobilizer” are less well-defined phenotypes than the “healthy” and “exhausted”, as evidence from the uneven spatial distribution and a high p value. Importantly, SOM allowed description of relevant clinical associations in the whole study cohort and is independent of the method of patients’ partitioning (the 40°/60° percentile rule).
DPP-4 in the diabetic stem cell mobilopathy
We tested the plasma concentration of a selected range of regulators of angiogenesis to evaluate whether our stem cell compartmentalization analysis is a suitable benchmark for the discovery of stem cell mobilizing factors. Plausible associations were indeed found such as between the CD34+CD133+KDR+ EPCs “healthy” and “good mobilizer” phenotypes and high concentrations of the mobilizing chemokine SDF-1α. An in-depth analysis of the plasma proteome could allow the identification of new mobilizing factors.
In addition, we found low activity of DPP-4 in the CD34+ “healthy” phenotype and high activity in patients with disturbed stem cell compartmentalization and mobilization. As peripheral DPP-4 activity is increased in DM , we performed pre-clinical experiments to validate causality of this association in the defective stem cell mobilization observed in DM. We employed two assays of stem/progenitor cell mobilization because the molecular mechanisms involved are different and may be differently affected by DPP-4. First, we confirm that both stimuli fail to mobilize hematopoietic (Sca-1+c-Kit+) and endothelial (Sca-1+CD31+) progenitor cells in diabetic rats, lending further support to the diabetic bone marrow failure. There are important implications of these findings, as BM stem/progenitor cell mobilization after ischemia is a physiologic response, while responsiveness to pharmacologic stimulation could be used therapeutically . The phenotypes used to define rat and human progenitor cells differ because there is no rat homology of human CD34 and CD133. Moreover, Sca-1+c-Kit+ cells contain hematopoietic progenitor cells in mice and rats, while co-expression of the endothelial antigen CD31 on Sca-1+ cells is compatible with a EPC phenotype . In addition, previous studies have found that these rat progenitor cell phenotypes are responsive to mobilizing stimuli [6, 20]. Interestingly, DPP-4 deficiency restored ischemia-induced progenitor cell mobilization in diabetic rats, reduced the degree of ischemia and preserved from the detrimental effects of diabetes on microvascular recovery. These data suggest that DPP-4 deficiency improves the outcome of ischemic tissues, although the number of capillaries in genetically deficient mice might be influenced by the resulting phenotype and the link with progenitor cell mobilization is indirect.
We have previously demonstrated that impaired post-ischemic progenitor cell mobilization in diabetic rats is attributable to an altered activation of the hypoxia-sensing system HIF-1α leading to a blunted release of the chemokine SDF-1α, a circulating mobilizing stimulus . As SDF-1α is a natural substrate of DPP-4 and is increased in diabetic patients after treatment with a DPP-4 inhibitor , excess DPP-4 activity is, therefore, one mechanism for reduced of SDF-1α in diabetes. Thus, we hypothesize that restored peripheral SDF-1α concentrations in DPP4null rats preserved the ability to mobilize BM progenitors. The measure of rat intact versus cleaved SDF-1α is challenging because traditional sandwich ELISA poorly discriminate between the two forms. However, proof-of-concept mass spectrometry analysis of mouse tissues confirmed that absence of DPP-4 reduces cleavage of native SDF-1α .
We have conducted a reverse translational study that stems from the observation of the clinical phenotype in patients with disturbed progenitor cell mobilization and leads to the discovery of a central role for DPP-4 in the diabetic stem cell mobilopathy.
In this clinical data-driven discovery approach for regenerative medicine, we used a targeted strategy to screen soluble factors potentially involved in progenitor cell regulation, but future development into the human plasma proteome analysis may allow an unbiased approach.
The tissue-specific regulation of DPP-4 in diabetes sheds light on the different mechanisms of ischemia- and GCSF-induced mobilization and has therapeutic implications based on clinically available DPP-4 inhibitors .
This work was supported by a European Foundation for the Study of Diabetes (EFSD)/Lilly Fellowship grant to GPF. There are no relationships with industry.
Conflict of interest