DPP-4 inhibition improves function of endothelial progenitor cells from type 2 diabetic patients

Background and aims. Type 2 diabetes (T2D) is associated with reduction and dysfunction of circulating pro-angiogenic cells (PACs). PACs are a subset of BM-derived cells that contribute to endothelial homeostasis. Diabetes impairs EPC functions, thus prompting endothelial dysfunction and cardiovascular diseases. DPP-4 inhibitors, such as Saxagliptin, are a relatively new class of antidiabetic agents. Since SDF-1α, a chemokine involved in EPC trafficking, is a substrate of DPP-4, we aimed to test whether DPP-4 inhibition affects basal and SDF-1α -stimulated EPC function Methods. PACs were isolated from T2D (n=20) and healthy (n=20) subjects. Gene expression, clonogenesis, proliferation, adhesion, migration and tubulisation were assessed in vitro by incubating PACs with or without Saxagliptin and SDF-1α. Stimulation of angiogenesis by circulating cells from T2D patients treated with Saxagliptin or other non-incretinergic drugs was assessed in vivo using animal models. Soluble DPP-4 activity was predominant over cellular activity and was successfully inhibited by Saxagliptin. At baseline, PACs from T2D patients compared to healthy PACs contained less acLDL+Lectin+ cells, and showed altered expression of genes related to adhesion and cell cycle regulation. This was reflected by impaired adhesion and clonogenesis / proliferative response of T2D PACs. Saxagliptin + SDF-1α improved adhesion and tube sustaining capacity of PACs from T2D patients, while it did not affect healthy PACs. While Saxagliptin modestly reduced angiogenesis by mature endothelial cells, circulating PAC-progeny cells from T2D patients on Saxagliptin treatment displayed higher growth factor-inducible in vivo angiogenetic activity, compared to cells from T2D patients on non-incretinergic regimen. Conclusions. Saxagliptin reverses PAC dysfunction associated with T2D in vitro and improves inducible angiogenesis by circulating cells in vivo. These data add knowledge to the potential pleiotropic cardiovascular effects of DPP-4 inhibition

Diabetes Causes Bone Marrow Autonomic Neuropathy and Impairs Stem Cell Mobilization via Dysregulated p66Shc and Sirt1

Diabetes compromises the bone marrow (BM) microenvironment and reduces circulating CD34 + cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system (SNS) is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34 + cells. Experimental diabetes (STZ and Ob/Ob ) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin - cKit + Sca1 + (LKS) cell and endothelial progenitor cells (EPC, CD34 + Flk1 + ) mobilization and vascular recovery after ischemia. DAN increased expression of p66Shc and reduced expression of Sirt1 in mice and humans. p66Shc KO in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, while hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1 , diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L . CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the lifespan regulators p66Shc and Sirt1

DPP-4 inhibition improves function of endothelial progenitor cells from type 2 diabetic patients

Background and aims. Type 2 diabetes (T2D) is associated with reduction and dysfunction of circulating pro-angiogenic cells (PACs). PACs are a subset of BM-derived cells that contribute to endothelial homeostasis. Diabetes impairs EPC functions, thus prompting endothelial dysfunction and cardiovascular diseases. DPP-4 inhibitors, such as Saxagliptin, are a relatively new class of antidiabetic agents. Since SDF-1α, a chemokine involved in EPC trafficking, is a substrate of DPP-4, we aimed to test whether DPP-4 inhibition affects basal and SDF-1α -stimulated EPC function Methods. PACs were isolated from T2D (n=20) and healthy (n=20) subjects. Gene expression, clonogenesis, proliferation, adhesion, migration and tubulisation were assessed in vitro by incubating PACs with or without Saxagliptin and SDF-1α. Stimulation of angiogenesis by circulating cells from T2D patients treated with Saxagliptin or other non-incretinergic drugs was assessed in vivo using animal models. Soluble DPP-4 activity was predominant over cellular activity and was successfully inhibited by Saxagliptin. At baseline, PACs from T2D patients compared to healthy PACs contained less acLDL+Lectin+ cells, and showed altered expression of genes related to adhesion and cell cycle regulation. This was reflected by impaired adhesion and clonogenesis / proliferative response of T2D PACs. Saxagliptin + SDF-1α improved adhesion and tube sustaining capacity of PACs from T2D patients, while it did not affect healthy PACs. While Saxagliptin modestly reduced angiogenesis by mature endothelial cells, circulating PAC-progeny cells from T2D patients on Saxagliptin treatment displayed higher growth factor-inducible in vivo angiogenetic activity, compared to cells from T2D patients on non-incretinergic regimen. Conclusions. Saxagliptin reverses PAC dysfunction associated with T2D in vitro and improves inducible angiogenesis by circulating cells in vivo. These data add knowledge to the potential pleiotropic cardiovascular effects of DPP-4 inhibition.Presupposti e obiettivi. Il diabete mellito (DM) è associato con una alterazione delle cellule pro angiogeniche (PACs). Le PACs sono cellule di origine midollare che contribuiscono all’omeostasi endoteliale. Il diabete altera le funzioni delle PACs favorendo quindi la disfunzione endoteliale. Gli inibitori dell’enzima DPP4, tra cui Saxagliptin (SAXA), sono una classe di farmaci usati nella terapia orale antidiabetica del diabete tipo 2. Poichè SDF-1α, chemochina coinvolta nella mobilizzazione delle PACs, è un substrato di DPP4, lo scopo dello studio è stato testare se l’inibizione di DPP4 possa modificare le capacità funzionali delle PACs. Metodi. Le PACs sono state isolate da sangue periferico di soggetti sani o diabetici e dopo sette giorni di coltura, in terreno addizionato di SAXA e/o SDF-1α, sono stati eseguiti alcuni saggi funzionali: adesione ad un monostrato di HUVECs, migrazione, proliferazione, tubulizzazione e analisi di espressione genica . Risultati. L’attività solubile di DPP-4 risulta maggiore rispetto all’attività della forma cellulare ed entrambe vengono equamente inibite da Saxagliptin. Al basale le colture di PACs da pazienti diabetici rispetto a quelle da soggetti sani contengono meno acLDL+Lectin+ cells e mostrano un’espressione alterata dei geni legati all’adesione e alla regolazione del ciclo cellulare. Questo rispecchia l’alterata risposta delle PACs da diabetici nei saggi di adesione, proliferazione / clono genesi. Saxagliptin + SDF-1α migliora l’adesione e la formazione di tubuli da parte delle PACs da soggetti diabetici ma non in quelle da soggetti sani. Le cellule da pazienti diabetici che assumono Saxagliptin mostrano una maggiore attivita angiogenica in vivo indotta da fattori di crescita rispetto a quelle da pazienti che non assumono Saxagliptin. Conclusioni. Saxagliptin ripristina la funzione delle PACs nel diabete in vitro e migliora l’angiogenesi indotta in vivo. Questi dati suggeriscono che l’inibizione di DPP4 possa avere un effetto positivo sulle PACs di pazienti diabetici, che potrebbero stimolare la rigenerazione endoteliale e ridurre il rischio di malattia cardiovascolare

Diabetes Impairs Stem Cell and Proangiogenic Cell Mobilization in Humans.

OBJECTIVE: Diabetes mellitus (DM) increases cardiovascular risk, at least in part, through shortage of vascular regenerative cells derived from the bone marrow (BM). In experimental models, DM causes morphological and functional BM alterations, but information on BM function in human DM is missing. Herein, we sought to assay mobilization of stem and proangiogenic cells in subjects with and without DM. RESEARCH DESIGN AND METHODS: In a prospective trial (NCT01102699), we tested BM responsiveness to 5 μg/kg human recombinant granulocyte colony–stimulating factor (hrG-CSF) in 24 individuals with DM (10 type 1 and 14 type 2) and 14 individuals without DM. Before and 24 h after hrG-CSF, we quantified circulating stem/progenitor cells and total and differential white blood cell counts. We also evaluated in vivo the proangiogenic capacity of peripheral blood mononuclear cells using the Matrigel plug assay. RESULTS: In response to hrG-CSF, levels of CD34(+) cells and other progenitor cell phenotypes increased in subjects without DM. Patients with DM had significantly impaired mobilization of CD34(+), CD133(+), and CD34(+)CD133(+) hematopoietic stem cells and CD133(+)KDR(+) endothelial progenitors, independently of potential confounders. The in vivo angiogenic capacity of peripheral blood mononuclear cells significantly increased after hrG-CSF in control subjects without DM, but not in patients with DM. DM was also associated with the inability to upregulate CD26/DPP-4 on CD34(+) cells, which is required for the mobilizing effect of granulocyte colony–stimulating factor. CONCLUSIONS: Stem and proangiogenic cell mobilization in response to hrG-CSF is impaired in DM, possibly because of maladaptive CD26/DPP-4 regulation. These alterations may hamper tissue repair and favor the development of cardiovascular complications

The molecular signature of impaired diabetic wound healing identifies serpinB3 as a healing biomarker

Aims/hypothesis Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The mechanisms underlying delaying wound healing in diabetes are incompletely understood and tools to identify such pathways are eagerly awaited. Methods Wound biopsies were obtained from 75 patients with diabetic foot ulcers. Matched subgroups of rapidly healing (RH, n = 17) and non-healing (NH, n = 11) patients were selected. Proteomic analysis was performed by labelling with isobaric tag for relative and absolute quantification and mass spectrometry. Differentially expressed proteins were analysed in NH vs RH for identification of pathogenic pathways. Individual sample gene/protein validation and in vivo validation of candidate pathways in mouse models were carried out. Results Pathway analyses were conducted on 92/286 proteins that were differentially expressed in NH vs RH. The following pathways were enriched in NH vs RH patients: apoptosis, protease inhibitors, epithelial differentiation, serine endopeptidase activity, coagulation and regulation of defence response. SerpinB3 was strongly upregulated in RH vs NH wounds, validated as protein and mRNA in individual samples. To test the relevance of serpinB3 in vivo, we used a transgenic mouse model with alpha 1-antitrypsin promoter-driven overexpression of human SERPINB3. In this model, wound healing was unaffected by SERPINB3 overexpression in non-diabetic or diabetic mice with or without hindlimb ischaemia. In an independent validation cohort of 47 patients, high serpinB3 protein content was confirmed as a biomarker of healing improvement. Conclusions/interpretation We provide a benchmark for the unbiased discovery of novel molecular targets and biomarkers of impaired diabetic wound healing. High serpinB3 protein content was found to be a biomarker of successful healing in diabetic patient