Dottorato in MEDICINA MOLECOLARE

Titolo della tesi: Pharmacological Intervention To Target The Pro-Calcific Phenotypic Drift Of Myeloid Cells: A Pilot Study

Introduction: Vascular calcification is prevalent in T2D (T2D) and is closely related to cardiovascular disease. Myeloid calcifying cells (MCCs) are monocytes with an osteoblast-like phenotype expressing osteocalcin (CD14+/OCN+). MCCs have been shown to contribute to cardiovascular disease (CVD) and vascular calcification in diabetes. Several clinic trials demonstrated that metformin, GLP-1 RA and pioglitazone all provide cardiovascular benefits, but mechanisms of CVD protection have been explained only in part. Thus, we hypothesized that metformin, GLP1-RA and pioglitazone can prevent the differentiation of monocytes towards an osteoblast-like phenotype and can reduce the levels of circulating MCCs, which have been identified as a new mechanism of cardiovascular injury, in diabetes. Study aims: To evaluate the effects of GLP1-RA added on metformin on the levels of MCCs in subjects affected by T2D. We also evaluated in vitro the effects of metformin and pioglitazone on the acquisition of an osteoblast-like (CD14+/OCN+) phenotype of cultured human monocytes. Materials and methods: In this prospective observational study, people with T2D on metformin who started add-on therapy with GLP1-RA (Group A) and people continuing treatment with metformin alone (Group B) were consecutively enrolled at the Diabetes Unit of Umberto I “Policlinico” General Hospital. CD14+/OCN+ MCCs levels were evaluate by flow cytometry before treatment, then one week and three months after treatment. Clinical and biochemical data were collected at baseline and on follow up from Electronic Health Records. Flow-cytometry assay was also used to detect surface expression of osteocalcin of cultured THP-1 monocytes treated for 48 hours with oxidized LDL (oxLDL) alone, or with pioglitazone ± oxLDL, or with metformin ± oxLDL at 48 hours. Results: Forty-eight have been enrolled, and flow cytometry analyses have been completed for 42 participants (10 in Group A and 34 in Group B). The overall cohort had a median [25th-75th percentiles] age of 64 [55.8-71.3] years, the HbA1c was 6.4 [6-6.8]%, and BMI was 29.3 [26.4-34.9] kg/m2. In both groups, a significant reduction in median [25th-75th percentile] MCC CD14+/OCN+ levels was observed already after one treatment week (Group A: 6.4% [1.9; 11.3] at baseline vs 1.8% [1.5; 3.5], p=0.028; Group B: 8.3% [4.5 ; 19.22] at baseline vs 8.2% [4.1; 10.9], p=0.013). The reduction in MCC CD14+/OCN+ levels was significantly higher in the group treated with GLP1-RA compared to metformin alone (p= 0.032). At three-months follow-up 6 participants within Group A and 25 participants within Group B completed the follow-up. The levels of CD14+OCN+ cells tended to be unchanged following metformin treatment during the three-month observation period, whereas they dropped significantly in the GLP1-RA add-on metformin treatment arm. Treatment with oxLDL increased OCN expression by 3-to 10-fold (p<0.05). Metformin ± oxLDL led to a non-statistically significant OCN expression reduction, whereas in vitro treatment with pioglitazone led to a significant reduction in OCN expression on THP-1 cells both alone (p=0.03) and after stimulation with oxLDL (p<0.001). Conclusions: These preliminary study results suggest that both metformin and GLP-1 RA could significantly decrease the circulating levels of CD14+/OCN+ MCCs, with a greater efficacy of GLP1-RAs, consistently with the known cardiovascular benefits of this class of drugs. The in vitro data also suggest possible benefits of pioglitazone in preventing the trans-differentiation of monocytes towards a calcifying phenotype, suggesting a new mechanism of cardiovascular action of these classes of drugs, widely used in people with T2D.