Diabetic retinopathy:The role of lipoprotein(a) and monocyte-derived cells in pathological angiogenesis

Diabetic retinopathy (DR) is a serious complication of type-2 diabetes mellitus (T2DM) and represents a major global cause of vision loss and blindness. Despite its clinical severity, there is a paucity of effective treatment for this condition. The current understanding of the pathogenesis of DR is insufficient, with gaps in knowledge and limitations in our comprehension of the underlying mechanisms involved. It is crucial to elucidate how inflammatory, vascular, and metabolic mechanisms interact in the complex pathogenesis of DR, and then implement this knowledge for development of innovative therapeutic advances. In order to bridge gaps in our understanding of DR pathogenesis, this thesis aims to address some fundamental aspects related to the microvascular complications associated with DR. Specifically, the role of lipoprotein(a) in the advancement of DR is a subject of debate, with limited exploration into its effects on the progression of DR and its associated microvascular complications. Moreover, the involvement and contribution of monocyte-derived cells to the progression of DR remain poorly understood. In particular, the pro-angiogenic properties of polarized macrophages and monocyte-derived pro-angiogenic cells (PAC) and their functional alterations under hyperglycemic conditions are investigated.The findings from this thesis showed that Lp(a) from patients with DR fail to inhibit inflammatory responses in retinal endothelial cells (REC), as compared to HC Lp(a). DR Lp(a) also stimulated REC in vitro angiogenesis while HC Lp(a) did not. Additionally, the phenotype of PAC, developing from healthy control monocytes, was altered in the presence of T2DM Lp(a), with less inhibition of PAC markers CD105 and CD133, as compared to the PAC generated with HC Lp(a).In search of a putative reflection of these functional differences in lipoprotein composition, we found that the lipid and protein profiles of Lp(a) and LDL from T2DM patients showed remarkable compositional differences compared to those from healthy individuals, particularly in terms of phospholipid content and complement factors.This thesis also showed that PAC formation is hampered in patients with T2DM, and that PAC from T2DM patients stimulate REC in vitro angiogenesis similarly compared to healthy PAC. Nevertheless, the cytokine profiles of the different PAC sources appeared to be different as T2DM PAC showed significantly higher production of S100A8, and lower secretion of VEGF-A. Together, these findings shed some light on the pro-inflammatory nature of T2DM PAC and their potential role in diabetic vascular dysfunction and retinopathy. This thesis also explores the phenotypic characteristics and angiogenic properties of different types of polarized macrophages under normo- and hyperglycemic conditions, thus in the context of DR. The findings indicate that, although the classically and alternatively activated macrophages had distinct phenotypic characteristics, they all had a mixed expression profile of pro- and anti-angiogenic factors. Hyperglycemia did not significantly affect the angiogenic properties of macrophages. It is concluded that the angiogenic status of polarized macrophages is not limited to a specific phenotype but is influenced by microenvironmental cues, leading to diverse phenotypic characteristics with variable implication for their angiogenic functionality.<br/

Diabetic retinopathy:The role of lipoprotein(a) and monocyte-derived cells in pathological angiogenesis

Diabetic retinopathy (DR) is a serious complication of type-2 diabetes mellitus (T2DM) and represents a major global cause of vision loss and blindness. Despite its clinical severity, there is a paucity of effective treatment for this condition. The current understanding of the pathogenesis of DR is insufficient, with gaps in knowledge and limitations in our comprehension of the underlying mechanisms involved. It is crucial to elucidate how inflammatory, vascular, and metabolic mechanisms interact in the complex pathogenesis of DR, and then implement this knowledge for development of innovative therapeutic advances. In order to bridge gaps in our understanding of DR pathogenesis, this thesis aims to address some fundamental aspects related to the microvascular complications associated with DR. Specifically, the role of lipoprotein(a) in the advancement of DR is a subject of debate, with limited exploration into its effects on the progression of DR and its associated microvascular complications. Moreover, the involvement and contribution of monocyte-derived cells to the progression of DR remain poorly understood. In particular, the pro-angiogenic properties of polarized macrophages and monocyte-derived pro-angiogenic cells (PAC) and their functional alterations under hyperglycemic conditions are investigated.The findings from this thesis showed that Lp(a) from patients with DR fail to inhibit inflammatory responses in retinal endothelial cells (REC), as compared to HC Lp(a). DR Lp(a) also stimulated REC in vitro angiogenesis while HC Lp(a) did not. Additionally, the phenotype of PAC, developing from healthy control monocytes, was altered in the presence of T2DM Lp(a), with less inhibition of PAC markers CD105 and CD133, as compared to the PAC generated with HC Lp(a).In search of a putative reflection of these functional differences in lipoprotein composition, we found that the lipid and protein profiles of Lp(a) and LDL from T2DM patients showed remarkable compositional differences compared to those from healthy individuals, particularly in terms of phospholipid content and complement factors.This thesis also showed that PAC formation is hampered in patients with T2DM, and that PAC from T2DM patients stimulate REC in vitro angiogenesis similarly compared to healthy PAC. Nevertheless, the cytokine profiles of the different PAC sources appeared to be different as T2DM PAC showed significantly higher production of S100A8, and lower secretion of VEGF-A. Together, these findings shed some light on the pro-inflammatory nature of T2DM PAC and their potential role in diabetic vascular dysfunction and retinopathy. This thesis also explores the phenotypic characteristics and angiogenic properties of different types of polarized macrophages under normo- and hyperglycemic conditions, thus in the context of DR. The findings indicate that, although the classically and alternatively activated macrophages had distinct phenotypic characteristics, they all had a mixed expression profile of pro- and anti-angiogenic factors. Hyperglycemia did not significantly affect the angiogenic properties of macrophages. It is concluded that the angiogenic status of polarized macrophages is not limited to a specific phenotype but is influenced by microenvironmental cues, leading to diverse phenotypic characteristics with variable implication for their angiogenic functionality.<br/

Caspase-Mediated Apoptosis in Sensory Neurons of Cultured Dorsal Root Ganglia in Adult Mouse

Objective: Sensory neurons in dorsal root ganglia (DRG) undergo apoptosis after peripheral nerve injury. The aim of this study was to investigate sensory neuron death and the mechanism involved in the death of these neurons in cultured DRG.Materials and Methods: In this experimental study, L5 DRG from adult mouse were dissected and incubated in culture medium for 24, 48, 72 and 96 hours. Freshly dissected and cultured DRG were then fixed and sectioned using a cryostat. Morphological and biochemical features of apoptosis were investigated using fluorescent staining (Propidium iodide and Hoechst 33342) and the terminal Deoxynucleotide transferase dUTP nick end labeling (TUNEL) method respectively. To study the role of caspases, general caspase inhibitor (Z-VAD.fmk, 100 μM) and immunohistochemistry for activated caspase-3 were used.Results: After 24, 48, 72 and 96 hours in culture, sensory neurons not only displayed morphological features of apoptosis but also they appeared TUNEL positive. The application of Z-VAD.fmk inhibited apoptosis in these neurons over the same time period. In addition, intense activated caspase-3 immunoreactivity was found both in the cytoplasm and the nuclei of these neurons after 24 and 48 hours.Conclusion: Results of the present study show caspase-dependent apoptosis in the sensory neurons of cultured DRG from adult mouse