What is the impact of diabetes and metabolic diseases on stem cell kinetics?
Which are the consequences of an altered bone marrow stem cell biology in diabetes?
Diabetes is the most common metabolic disease and it shortens life expectancy owing to multi-origan chronic complications affecting the eyes, kidneys, nerves, heart and vessels. We are interested in the mechanisms and consequences of bone marrow structural and functional alterations in diabetes. The bone marrow contains stem cells and is site of hematopoiesis. Diabetes compromises stem cell mobilization and alters the phenotype of several leukocytes types. Understanding the causes and clinical implications of these changes is our challenge.
Diabetes is progressively growing worldwide with epidemic diffusion. Its prevalence reaches up to 10% of the general population in Western countries, and it is growing even faster in the developing world. Importantly, diabetes leads to severe multi-organ complications and is associated with a shortened life expectancy. Diabetes develops as a consequence of a complex interplay between genes and environment. The metabolic alterations observed in diabetes promote the development of chronic complications within several organs, including the arteries, heart, kidneys, nerves and eyes. In turn, these are responsible for the high burden of morbidity and mortality in diabetic patients. In the Laboratory of Experimental Diabetology, research activities are focused on the cellular and molecular mechanisms underlying the development of diabetes and its long-term complications, as well as the therapeutic strategies to prevent and treat these conditions. In the Lab, expertise from multiple disciplines converge to increase the impact of the work, including clinical medicine and basic science.
The specific commitment of this Lab is to integrate basic science with clinical medicine, creating a unique environment, ideal to provide significant advancements in the field, through approaches of “translational medicine”. The group comprises a set of medical doctors, research fellows, as well as lab personnel with extensive experience in molecular and cellular biology, in silico studies, tissue analysis, and animal models of disease. Our major goal is to study how the bone marrow and stem cells are affected by diabetes and contribute to the development of metabolic abnormalities and chronic complications.
Therapeutic modulation of stem and progenitor cells in diabetes to interrupt the pathologic processes leading to complications
We have extensively characterized alterations of bone marrow-derived stem / progenitor cells in vitro as well as in vivo in animals and in humans with diabetes. Thanks to the contribution of our lab, endothelial progenitor cell (EPCs) are now considered a determinant of vascular health and their alterations in the setting of diabetes a driver of complications. We have established a connection between the number and function of EPCs to the development of long-term macro- and microvascular complications. These studies have allowed to define a new model that emphasizes the crucial role of the progressive shortage of bone-marrow derived stem cells, a condition similar to that observed in the ageing process. We have also identified pharmacologic approaches able to restore the stem cell population, which are being transferred to the clinical setting (Figure 1).
Future efforts are devoted to the study of further potential molecular mechanisms and therapeutic implications. The use of animal models will allow to asses the regulation of vascular progenitor cells in the presence of diabetes. Application of our knowledge on progenitor cell shortage in diabetes is close to identification of clinical application and therapies.
Figure 1. Origin and function of circulating (progenitor) cell phenotypes in diabetic complications. While the bone marrow haemangioblast is considered the origin of classical true EPCs, ECFC are believed to derive from the vessel wall. Early EPCs (more properly renamed as CACs or PACs), smooth muscle progenitor cells (SMPCs) and M1/M2 phenotype originate from the monocyte/macrophage lineage. Red symbols indicate quantitative changes in the various cells types. ED, endothelial dysfunction; IMT, intima-media thickening. Modified from Fadini GP, Diabetologia 2014.
The diabetic bone marrow as a link to distant end-organ complications.
We have moved from the analysis of definite progenitor cell phenotypes and their characteristics to the study of bone marrow alterations induced by diabetes. Ee have clarified that diabetes is characterized by a dysfunction of the stem cell niche, which causes a significant impairment in stem cell mobilization from the bone marrow into peripheral blood. Besides its importance in the haematology field, this new notion has also strong implications for the development and progression of diabetic complications, because bone marrow derived cells contribute to the homeostasis of non-hematopoietic organs. More specifically, we found that diabetes-induced impairment in stem cell mobilization after ischemia and G-CSF depend, at least in part, on a tissue specific dysregulation of DPP-4 activity. In mice, we also found that diabetes induced sympathetic and sensory denervation of the bone marrow, which is critical for the induction of diabetic stem cell mobilopathy. The longevity gene and adaptor protein p66Shc mediates bone marrow denervation, as p66Shc KO mice are protected from BM denervation and restores mobilization. Furthermore, the mechanisms behind diabetes and sympathectomy induced mobilopathy relies on cell-intrinsic pathways involving Sirtuin-1 and adhesion molecules. Genetic manipulation of Sirt-1 and L-selectin are indeed able to restore stem cell mobilization in diabetic and sympathectomised mice. In parallel to bone marrow sympathectomy, we found that inflammation drives myelopoiesis and differentiation of intramarrow macrophages that, when presence in excess such as in diabetes, retain stem cells in the niche by producing oncostatin M (OSM).
Figure 2. The mechanisms of EPC alterations in diabetes. A 3 compartment model is shown in which EPC derive from the bone marrow, are mobilized to the bloodstream and home to peripheral target tissues. Hyperglycemic damage pathways along with inflammation and oxidative stress contribute to the development of bone marrow microangiopathy and neuropathy which, in turn, impair EPC mobilization. Once in the diabetic circulation, EPCs are subjected to a series of molecular challenges and their survival can also be impaired by subtle inflammation and reactive oxygen species. The homing signal involving SDF-1 and DPP-4 are defective in diabetes, but homing to peripheral tissues is area of interest as several other mechanisms may be involved (modified from Menegazzo et al. Biofactors 2012).
Understanding the origin of vascular calcification in diabetes through the contribution of circulating cells
We have identified in mice and humans a novel subpopulation of monocyte/macrophages expressing the bone-related proteins osteocalcin (OC) and alkaline phosphatase (BAP), which contribute to ectopic calcification and are over-represented in diabetes. Such so-called myeloid calcifying cells (MCC) can be viewed in the framework of monocyte plasticity and are expected to be involved in several physiologic and pathologic processes. In humans, MCC are increased in the bloodstream, bone marrow, and atherosclerotic plaques of diabetic compared to non-diabetic patients and retain the capacity to calcify in vitro and in vivo. This is in line with the well-known excess of vascular calcification, which is typical of diabetes. In mice, an adoptive cell transfer strategy has demonstrated that MCC worsen atherosclerotic calcification in ApoE-/- mice mainly through paracrine activity and likely via an overexpression of the macrophage activating factor allograft inflammatory factor-1 (AIF-1). In addition, we have recently found that human MCC are endowed with anti-angiogenic activity in vitro and in vivo, by means of both cell-intrinsic and paracrine activity through hyperproduction of the anti-angiogenic factor thrombospondin-1 (TSP-1). Induction of calcification and inhibition of angiogenesis are 2 important functions of MCCs that make them major candidate mechanisms of the peculiar characteristics of diabetic vasculopathy (Figure 3).
Figure 3. Hypothetical pathophysiological implications of myeloid calcifying cells in atherosclerotic diseases, through induction of calcification and inhibition of angiogenesis.
NETosis as a link between the environment, metabolism and diabetic complications
Diabetes and the metabolic syndrome are characterized by activation of the innate immune system. As the lab is committed to the study of circulating cells in diabetes, we have recently moved to analyse the role played by neutrophils. Upon challenge with microbes and inflammatory triggers, neutrophils undergo histone citrullination by protein arginine deiminase-4 (PAD4), release enzymes and nuclear material, forming neutrophils extracellular traps (NETs) and thereby dying by NETosis. We have found for the first time that hyperglycemia increase release of NETs and circulating markers of NETosis. This finding provides a link among neutrophils, inflammation and tissue damage in diabetes. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFU). Using proteomics, we found that NET components were enriched in non-healing human DFU. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components were elevated in the blood of patients with DFU. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced netting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing in mice and humans.
In diabetes, the finely tuned balance of NETosis required to protect the human body from microorganisms yet avoiding self-damage seems to be lost. Furthermore, NETs contribute to endothelial damage, thrombosis, and ischemia/reperfusion injury, making it a novel player in the pathobiology of cardiovascular disease (Figure 4).
Figure 4. An overview of the pathophysiology of NETosis. Pathological changes in the oral or gut microbiome can stimulate NETosis. This has been demonstrated for periodontitis and is speculative for what concerns the gut microbiome. Changes in microbiota, as well as tissue damage (such as cutaneous wounds) and infections, can induce local NETosis. Systemic NETosis can be the result of the spreading of bacterial products through the bloodstream. As shown in the exploded central box, the cascade of events taking place in NETosis include PAD4-mediated histone citrullination (1), followed by chromatin decondensation (2), disintegration of the nuclear envelope, enter of granule content into the nucleus and extrusion of nuclear material with enzymes and other granule proteins (3). These steps can also be easily shown by transmission electron microscopy (Fadini & Menegazzo, unpublished data), which recapitulates events described in Figure 1. As a result of NETosis, the concentration of NET components increase in the circulation, and they can promote adverse clinical and pathophysiological sequelae, as indicated in the boxes below (modified from Fadini et al. NMCD 2016).
Video 1. Live recording of NETosis in human isolated neutrophils. The video features a live recording of human neutrophils isolated using a non-activating immuno-magnetic cell sorting method. Recording starts 20 minutes after addition of PMA. Nuclei are stained in blue with the cell-permeant Hoechst 33342 dye; mitochondria are stained in red with tetramethylrhodamine methyl ester (TMRM), which labels only energized organelles because its signal is proportional to DeltaPsi; extracellular double strand (ds) DNA is stained with the cell-impermeant Sytox green, present in the medium. In the first part, the black arrow indicates a neutrophil with loss of mitochondrial energization, initial nuclear delobulation, and chromatin decondensation, whereas surrounding neutrophils still show normal polymorphonuclear shape and mitochondrial signals. In the second part, most cells have lost mitochondrial membrane potential and the cell identified earlier undergoes extensive chromatin decondensation, evidenced by the progressive nuclear expansion and tapering of the Hoechst 33342 signal (which is proportional to DNA concentration). This culminates in the release of dsDNA into the extracellular space, where is binds to Sytox green present in the medium, which becomes brightly fluorescent. The sticky dsDNA, complexed with enzymes released from neutrophil granules form neutrophil extracellular traps (NETs).
Future research plans
Study of the inter-relationships between bone marrow niche dysfunction and structural changes induced by diabetes in the bone marrow including adipocytes, the microvasculature and inflammatory cells.
Exploring potential therapeutic strategies in diabetes to restore vascular repair processes mediated by bone marrow-derived cells. To this end, we focus on pharmaceutical compounds that currently used in clinical practice or will become available in the years to come. Both studies in vitro and in vivo will be developed to assess the potential of these approaches to reverse diabetes-associated anomalies in progenitor cell levels and function.
We are exploring the roles of myeloid calcifying cells (MCCs) in atherosclerotic calcifications of diabetic patients in vivo, also in relation to other potential mechanisms of vascular calcification induced by soluble mediators and cell-related processes.
We are studying novel molecular signatures of delayed diabetic wound healing, starting from data generated by unbiased proteomic and genomic approaches that will be validated using in vitro and in vivo approaches.
We are characterizing in detail the multiple alterations seen in neutrophils from patients and animals with diabetes, testing how NETosis is induced by diabetes and whether it is regulated by the gut microbiome, a potent mediator of inflammaotty and metabolic signals.
- Tedesco S, Ciciliot S, Menegazzo L, D'Anna M, Scattolini V, Cappellari R, Cignarella A, Avogaro A, Albiero M, Fadini GP. Pharmacologic PPAR-γ Activation Reprograms Bone Marrow Macrophages and Partially Rescues HSPC Mobilization in Human and Murine Diabetes. Diabetes. 2020 Apr 28
- Cappellari R, D'Anna M, Menegazzo L, Bonora BM, Albiero M, Avogaro A, Fadini GP. Diabetes mellitus impairs circulating proangiogenic granulocytes. Diabetologia. 2020 Apr 18.
- Fadini GP, Spinetti G, Santopaolo M, Madeddu P. Impaired Regeneration Contributes to Poor Outcomes in Diabetic Peripheral Artery Disease. Arterioscler Thromb Vasc Biol. 2020 Jan
- Bonora BM, Vigili de Kreutzenberg S, Avogaro A, Fadini GP. Effects of the SGLT2 inhibitor dapagliflozin on cardiac function evaluated by impedance cardiography in patients with type 2 diabetes. Secondary analysis of a randomized placebo-controlled trial. Cardiovasc Diabetol. 2019 Aug 14
- Fadini GP, Albiero M, Bonora BM, Avogaro A. Angiogenic Abnormalities in Diabetes Mellitus: Mechanistic and Clinical Aspects. J Clin Endocrinol Metab. 2019 Nov
- Tedesco S, Scattolini V, Albiero M, Bortolozzi M, Avogaro A, Cignarella A, Fadini GP. Mitochondrial Calcium Uptake Is Instrumental to Alternative Macrophage Polarization and Phagocytic Activity. Int J Mol Sci. 2019 Oct 8
- Fadini GP, Mehta A, Dhindsa DS, Bonora BM, Sreejit G, Nagareddy P, Quyyumi AA. Circulating stem cells and cardiovascular outcomes: from basic science to the clinic. Eur Heart J. 2019 Dec 31
- Albiero M, Ciciliot S, Tedesco S, Menegazzo L, D'Anna M, Scattolini V, Cappellari R, Zuccolotto G, Rosato A, Cignarella A, Giorgio M, Avogaro A, Fadini GP. Diabetes-Associated Myelopoiesis Drives Stem Cell Mobilopathy Through an OSM-p66Shc Signaling Pathway. Diabetes 2019 Apr 1.
- Gherardi G, Nogara L, Ciciliot S, Fadini GP, Blaauw B, Braghetta P, Bonaldo P, De Stefani D, Rizzuto R, Mammucari C. Loss of mitochondrial calcium uniporter rewires skeletal muscle metabolism and substrate preference. Cell Death Differ. 2019 Jan
- Menegazzo L, Scattolini V, Cappellari R, Bonora BM, Albiero M, Bortolozzi M, Romanato F, Ceolotto G, Vigili de Kreutzeberg S, Avogaro A, Fadini GP. The antidiabetic drug metformin blunts NETosis in vitro and reduces circulating NETosis biomarkers in vivo. Acta Diabetol. 2018;55:593-601.
- Ciciliot S, Albiero M, Campanaro S, Poncina N, Tedesco S, Scattolini V, Dalla Costa F, Cignarella A, Vettore M, Di Gangi IM, Bogialli S, Avogaro A, Fadini GP. Interplay between gut microbiota and p66Shc affects obesity-associated insulin resistance. FASEB J. 2018;32:4004-4015.
- Fadini GP, Albiero M, Bonora BM, Poncina N, Vigili de Kreutzenberg S, Avogaro A. p66Shc gene expression in peripheral blood mononuclear cells and progression of diabetic complications. Cardiovasc Diabetol. 2018 Jan 17;17(1):16.
- Fadini GP, Boscari F, Cappellari R, Galasso S, Rigato M, Bonora BM, D'Anna M, Bruttomesso D, Avogaro A. Effects of Hypoglycemia on Circulating Stem and Progenitor Cells in Diabetic Patients. J Clin Endocrinol Metab. 2018;103:1048-1055.
- Ben Nasr M, Tezza S, D'Addio F, Mameli C, Usuelli V, Maestroni A, Corradi D, Belletti S, Albarello L, Becchi G, Fadini GP, Schuetz C, Markmann J, Wasserfall C, Zon L, Zuccotti GV, Fiorina P. PD-L1 genetic overexpression or pharmacological restoration in hematopoietic stem and progenitor cells reverses autoimmune diabetes. Sci Transl Med. 2017 Nov 15;9(416).
- Rigato M, Avogaro A, Fadini GP. Levels of Circulating Progenitor Cells, Cardiovascular Outcomes and Death: A Meta-Analysis of Prospective Observational Studies. Circ Res. 2016 Jun 10;118(12):1930-9.
- Fadini GP, Menegazzo L, Rigato M, Scattolini V, Poncina N, Bruttocao A, Ciciliot S, Mammano F, Ciubotaru CD, Brocco E, Marescotti MC, Cappellari R, Arrigoni G, Millioni R, Vigili de Kreutzenberg S, Albiero M, Avogaro A. NETosis Delays Diabetic Wound Healing in Mice and Humans. Diabetes. 2016 Apr;65(4):1061-71.
- Fadini GP, Fiala M, Cappellari R, Danna M, Park S, Poncina N, Menegazzo L, Albiero M, DiPersio J, Stockerl-Goldstein K, Avogaro A. Diabetes Limits Stem Cell Mobilization Following G-CSF but Not Plerixafor. Diabetes. 2015 Aug;64(8):2969-77.
- Albiero M, Poncina N, Ciciliot S, Cappellari R, Menegazzo L, Ferraro F, Bolego C, Cignarella A, Avogaro A, Fadini GP. Bone Marrow Macrophages Contribute to Diabetic Stem Cell Mobilopathy by Producing Oncostatin Diabetes. 2015 Aug;64(8):2957-68.
- Albiero M, Poncina N, Tjwa M, Ciciliot S, Menegazzo L, Ceolotto G, Vigili de Kreutzenberg S, Moura R, Giorgio M, Pelicci P, Avogaro A, Fadini GP. Diabetes causes bone marrow autonomic neuropathy and impairs stem cell mobilization via dysregulated p66Shc and Sirt1. Diabetes. 2014 Apr;63(4):1353-65.:
- Menegazzo L, Albiero M, Millioni R, Tolin S, Arrigoni G, Poncina N, Tessari P, Avogaro A, Fadini GP. Circulating myeloid calcifying cells have antiangiogenic activity via thrombospondin-1 overexpression. FASEB J. 2013 Nov;27(11):4355-65.
- Fadini GP, Rattazzi M, Matsumoto T, Asahara T, Khosla S. Emerging role of circulating calcifying cells in the bone-vascular axis. Circulation. 2012 Jun 5;125(22):2772-81.
- Fadini GP, Albiero M, Menegazzo L, Boscaro E, Vigili de Kreutzenberg S, Agostini C, Cabrelle A, Binotto G, Rattazzi M, Bertacco E, Bertorelle R, Biasini L, Mion M, Plebani M, Ceolotto G, Angelini A, Castellani C, Menegolo M, Grego F, Dimmeler S, Seeger F, Zeiher A, Tiengo A, Avogaro A. Widespread increase in myeloid calcifying cells contributes to ectopic vascular calcification in type 2 diabetes. Circ Res. 2011 Apr 29;108(9):1112-21.
- Fadini GP, Albiero M, Menegazzo L, Boscaro E, Pagnin E, Iori E, Cosma C, Lapolla A, Pengo V, Stendardo M, Agostini C, Pelicci PG, Giorgio M, Avogaro A. The redox enzyme p66Shc contributes to diabetes and ischemia-induced delay in cutaneous wound healing. Diabetes. 2010 Sep;59(9):2306-14.
GIAN PAOLO FADINI
- MD: University of Padua (2004)
- PhD: University of Padua (2013)
- Group leader: Veneto Institute of Molecular Medicine, (2015-current)
- Assistant professor of Endocrinology, University of Padua, Department of Medicine (2010-2015)
- Associate professor of Endocrinology, University of Padua, Department of Medicine (2016-current)
- 2008 – Morgagni prize silver medal
- 2009 – EASD Rising Star
- 2014 – Italian Diabetes Society Alcmeone Prize
- 2015 – “2003 Group” prize