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2014-2015 - Relazione Progetto: Joslin Diabetes Center – Harvard Medcal School

La relazione finale del borsista Ernesto Maddaloni

In October 2014 I joined the laboratory of Vascular Cell Biology at the Joslin Diabetes Center (Harvard Medical School) for a one-year post-doctoral research fellowship under the mentorship of Prof. George L. King.  The Joslin Diabetes Center is the world's largest diabetes research and clinical care organization. It comprises a comprehensive and productive effort in diabetes research and it is a worldwide recognized leading center in diabetes research and management. Researches at the Joslin focus on unrevealing the biological, biochemical and genetic processes that underlie the development of type 1 and type 2 diabetes and related complications.

During my time at the Joslin Diabetes Center I acquired new skills in diverse fields including lab techniques, data collection and analyses, study design and research methods, combining them to perform translational research that is imperative in today’s scientific environment. In particular I had the opportunity to practice and learn:

  • Bio-statistic skills including parametric and non-paramentric tests of hypotheses, tests of normality, analysis of Variance and Covariance, higher order linear and logistic regression modeling, multilevel mixed-effects model
  • Western-blot
  • RNA extraction by TRIzol from tissues and cell cultures
  • Spin column-based nucleic acid purification from tissues and cell cultures
  • Polymerase Chain Reaction (PCR)
  • Cell cultures (primary human osteoblast culture, osteoblast isolation and cultures from murine long-bones, culture of human fibroblasts and monocytes)
  • Isolation of “Peripheral Blood Mononuclear Cells (PBMCs)” from human blood
  • Flow Citometry and Fluorescence Activated Cell Sorting

The main focus of the research project I conducted at the Joslin Diabetes Center has been bone fragility and its relationship with cardiovascular diseases in the setting of diabetes. In particular, we aimed to evaluate the role of circulating progenitor cells in the development of vascular complications of diabetes in the Joslin 50-Year Medalist cohort study, focusing on the ratio of circulating cells expressing markers of bone formation between Medalists with and without cardiovascular disease. The 50-year Medalist Study is an ongoing research project at the Joslin that involves a large number of individuals who have survived fifty or more years with type 1 diabetes. Thus, the Medalist population provides a unique opportunity to study individuals with extreme duration of diabetes. The Study is currently enrolling Medalists from within the United States in an effort to identify genetic, environmental, psychological and physiological factors, which may contribute to survival with extreme duration of diabetes. To date the Joslin 50-Year Medalist Study has identified and extensively characterized more than 1000 subjects who have survived with over 50 years of type 1 diabetes, offering insight on endogenous preventive factors from diabetic complications. However, the Medalists have never been studied in regards to bone health. Thus, during my fellowship at the Joslin I investigated:

  • Bone mineral density and bone quality in the Medalist and the eventual association between diabetic osteopathy and vascular complications of diabetes. We collected data about bone fractures in 985 Medalists (455 males and 530 females) showing an extraordinary low prevalence of fragility fractures, with congruent values of bone mineral density. This allows us to consider the 50-year Medalists as a unique model to search for factors that could protect T1D individuals from the development of bone fragility, which will be the focus of a future project funded by the NIH (https://www.diacomp.org/shared/showSubContractAbstract.aspx?id=2509). We also found a strong and independent association between CVD and femoral neck osteopenia. One novel aspect of this work has also been the finding of differential patterns of bone microarchitecture change across anatomical sites in Medalists compared to age-matched non-diabetic and type 2 diabetic controls. Part of these data have been accepted to be presented as late breaking abstract at the 2015 meeting of the American Society for Bone and Mineral Research (ASBMR) in Seattle, October 9-12.
     
  • The role of circulating progenitor cells in the development of diabetic complications, Analysis of circulating endothelial progenitor cells (EPC) in 172 Medalists showed that these cells are higher in subjects with estimated glomerular filtration rate (eGFR) >90ml/min/1.73m2 than in those with eGFR between 60-90 and <60  (151.0 ± 51.1 vs 132.6 ± 50.3 vs 126.5 ± 6.2 per 106 cells, p= 0.03). In normoalbuminuric subjects (n=133), EPC remained positively associated with eGFR after adjustment for gender, age, HbA1c, SDF1, sVCAM, cardiovascular disease and hypertension (Adj R2 0.29, p< 0.01). Overall, we found thathigh levels of circulating EPC are associated with lower plasma IGF-1 and preservation of eGFR in type 1 diabetes of chronic duration, suggesting this could be a biomarker, and possibly involved as a protective mechanisms against the development of DN. The full data where presented at the 75th meeting of the American Diabetes Association, published in Diabetes, Suppl. 1, Pag A148-149 and are available at http://app.core-apps.com/tristar_ada15/abstract/160858d53930b598d64b10f393cf7ba5.
     
  • Whether the differentiation of circulating cells into osteoblast-like cell) is involved in the relationship between cardiovascular disease and bone health (namely the “bone-vascular axis”). The data we have collected so far suggest an association between myeloid cells co-expressing a marker of bone formation, history of cardiovascular disease and risk factors for the development of atherosclerosis in subjects with extreme duration type 1 diabetes. Further in vitro and in vivo studies are currently ongoing to clarify the involvement of monocytes expressing osteoblast-specific markers in the development of atherosclerosis and to individuate factors involved in the induction of the differentiation of  monocytes into “osteoblast-like” cells.

Finally I actively participated in the ongoing research activities of the Vascular Cell Biology laboratory, in particular working on research projects aimed 1) to elucidate insulin signaling and actions in human induced pluripotent stem (hiPSC) cells derived from fibroblasts from type 1 diabetic patients (data presented at the 75th meeting of the American Diabetes Association, published in Diabetes, Suppl. 1, Pag A182 and are available at http://app.core-apps.com/tristar_ada15/abstract/160858d53930b598d64b10f3930f688a), and 2) to evaluate the role of endothelial specific insulin resistance in the pathogenesis of atherosclerosis in order to identify innovative targets to reverse this pathological process in diabetes.