Gian Paolo Fadini

LABORATORY OF EXPERIMENTAL DIABETOLOGY

 

Field of Interest

In the Laboratory of Experimental Diabetology, research activities are focused on the cellular and molecular mechanisms underlying the development of diabetes mellitus 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.
Diabetes is progressively growing worldwide with epidemic diffusion. Its prevalence reaches 7% 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. These contribute to the high burden of morbidity and mortality in diabetic patients.
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.

Ongoing research projects

• Mesenchymal, hematopoietic and vascular stem/progenitor cells in diabetes: evaluating possible therapeutic modulation of these cells to interrupt the pathologic processes leading to diabetic complications;

• Exploring alterations induced by diabetes onto the bone marrow structure and function, which is considered to be responsible of the defective tissue repair associated with diabetes and that contribute to development and progression of complications;

• Understanding the origin and mechanisms of excess vascular calcification in diabetes and the contribution of circulating cells to the pathobiology of diabetic vascular disease;

• Determining the pathophysiological contribution of exaggerated oxidative stress in the glucometabolic regulation and its role in the progression of macro- and microvascular complications, such as vascular disease and diabetic wounds;

• Defining the relationships between longevity-associated genes, metabolic disturbances, and long-term diabetic complications. To understand why diabetes shortens life span and how to circumvent the pathological processes leading to this sort of “precocious ageing”.

Significant results obtained

In the last 10 years our group has achieved important results in the following research areas.

• 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 assess the regulation of vascular progenitor cells in the presence of diabetes. We are currently moving to consider the biology of circulating stem/progenitor cells from a wider pathophysiological perspective, taking into account the potential of these cells in the cardiovascular system, through differentiation into pro-calcific cells and smooth muscle cells. 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. In addition 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.

• In the last years we are moving from the analysis of definite progenitor cell phenotypes and their characteristics to the study of bone marrow alterations induced by diabetes. In particular, we 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. This new notion, besides its importance in the hematology field, has also strong implications for the development and progression of diabetic complications, as 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. Interestingly, this can be modulated by DPP-4 inhibitors to restore ischemia induced mobilization of vascular repairing cells. 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, which are dysregulated. Genetic manipulation of Sirt-1 and L-selecting are indeed able to restore stem cell mobilization in diabetic and sympathectomised mice. We are currently exploring in more detail the interactions between niche dysfunction and structural changes in the bone marrow, including those related to marrow fat, vasculature and inflammatory cells.

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 bore 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).

 

 

 

• We have recently 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 mellitus. 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.

 

* 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.

 

 

GIAN PAOLO FADINI, MD PhD

 


2016-present: Associate Professor of Endocrinology at the University of Padova, Department of Medicine.

2010-2015: Assistant Professor of Endocrinology at the University of Padova, Department of Medicine.

2010-present: Associate Medical Director, Division of Metabolic Disease, University Hospital of Padova

2008: Training fellow in Molecular Cardiology, University of Frankfurt

2005-2009: Specialization in Endocrinology and Metabolism, University of Padova

2004: Degree in Medicine and Surgery, University of Padova.

Awards / Honors

2015: Young investigator award “Gruppo 2003 per la ricerca”

2014   “Alcmeone” prize of the Italian Society of Diabetology

2014   “Cornaro prize” for the study on aging

2011   “Angelo Minich” Award in Medicine

2010   “Young Investigator Award” of the Italian Society of Diabetology

2010   “Lilly Foundation publication” award in Endocrinology and Metabolism

2009   “Rising Star Symposium” award of the European Association for the Study of Diabetes (EASD)

2008   Morgagni Silver Medal for research on progenitor cells and diabetic complications

Fields of study

Acute and chronic vascular diabetic complications

Stem cells in diabetes

Angiogenesis

Atherosclerosis

Vascular calcification

Vascular regeneration

Wound healing

Metabolic syndrome and insulin resistance

Oxidative stress

Longevity gene pathways in metabolic diseases

Selected publications

 

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 Jan 6. pii:db150863.

Dang Z, Maselli D, Spinetti G, Sangalli E, Carnelli F, Rosa F, Seganfreddo E, Canal F, Furlan A, Paccagnella A, Paiola E, Lorusso B, Specchia C, Albiero M, Cappellari R, Avogaro A, Falco A, Quaini F, Ou K, Rodriguez-Arabaolaza I, Emanueli C, Sambataro M, Fadini GP, Madeddu P. Sensory neuropathy hampers nociception-mediated bone marrow stem cell release in mice and patients with diabetes. Diabetologia. 2015 Nov;58(11):2653-62.

Ciciliot S, Albiero M, Menegazzo L, Poncina N, Scattolini V, Danesi A, Pagnin E, Marabita M, Blaauw B, Giorgio M, Trinei M, Foletto M, Prevedello L, Nitti D, Avogaro A, Fadini GP. p66Shc deletion or deficiency protects from obesity but not metabolic dysfunction in mice and humans. Diabetologia. 2015 Oct;58(10):2352-60.

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 M. Diabetes. 2015 Aug;64(8):2957-68.

Fadini GP, Albiero M, Millioni R, Poncina N, Rigato M, Scotton R, Boscari F, Brocco E, Arrigoni G, Villano G, Turato C, Biasiolo A, Pontisso P, Avogaro A. The molecular signature of impaired diabetic wound healing identifies serpinB3 as a healing biomarker. Diabetologia. 2014 Sep;57(9):1947-56.

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, Albiero M, Vigili de Kreutzenberg S, Boscaro E, Cappellari R, Marescotti M, Poncina N, Agostini C, Avogaro A. Diabetes impairs stem cell and proangiogenic cell mobilization in humans. Diabetes Care. 2013 Apr;36(4):943-9.

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, Losordo D, Dimmeler S. Critical reevaluation of endothelial progenitor cell phenotypes for therapeutic and diagnostic use. Circ Res. 2012 Feb 17;110(4):624-37.

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 inmyeloid calcifying cells contributes to ectopic vascular calcification in type 2 diabetes. Circ Res. 2011 Apr 29;108(9):1112-21.

Albiero M, Menegazzo L, Boscaro E, Agostini C, Avogaro A, Fadini GP. Defective recruitment, survival and proliferation of bone marrow-derived progenitor cells at sites of delayed diabetic wound healing in mice. Diabetologia. 2011 Apr;54(4):945-53.

Fadini GP, Ceolotto G, Pagnin E, de Kreutzenberg S, Avogaro A. At the crossroads of longevity and metabolism: the metabolic syndrome and lifespan determinant pathways. Aging Cell. 2011 Feb;10(1):10-7.

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.

Fadini GP, Sartore S, Schiavon M, Albiero M, Baesso I, Cabrelle A, Agostini C, Avogaro A. Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia-reperfusion injury in rats. Diabetologia. 2006 Dec;49(12):3075-84.