Abstract
ABSTRACTTo understand the pathology and molecular signatures of microangiopathy in diabetic neuropathy, we systemically and quantitatively examined the morphometry of microvascular and nerve pathologies of sural nerves. In the endoneurium of diabetic nerves, prominent microangiopathy was observed, as evidenced by reduced capillary luminal area, increased capillary basement membrane thickness and increased proportion of fibrin(+) blood vessels. Furthermore, capillary basement membrane thickness and the proportion of fibrin(+) blood vessels were correlated with small myelinated fiber density in diabetic nerves. In diabetic nerves, there was also significant macrophage and T cell infiltration, and cluster of differentiation 40 (CD40) expression was increased. The molecular alterations observed were upregulation of hypoxia-inducible factor-1α (HIF-1α), mitogen-activated protein kinase-activated protein kinase 2 (MK2; MAPKAPK2) and phosphatase and tensin homolog (PTEN). In addition, HIF-1α was correlated with small myelinated fiber density and capillary luminal area, while both MK2 and PTEN were correlated with capillary basement membrane thickness. The molecular cascades were further demonstrated and replicated in a cell model of microangiopathy on human umbilical vein endothelial cells (HUVECs) exposed to high-glucose medium by silencing of CD40, PTEN and HIF-1α in HUVECs using shRNA. These data clarified the hierarchy of the molecular cascades, i.e. upregulation of CD40 leading to HIF-1α expression in endothelium and nerve fibers. In conclusion, this study revealed the association of microangiopathy, thrombosis and inflammatory infiltrates with nerve degeneration in diabetic nerves, demonstrating that CD40 is a key molecule for the upregulation of HIF-1α and PTEN underlying the severity of microangiopathy.
Highlights
Peripheral nerve degeneration in diabetic neuropathy, a major complication of diabetes with a frequency of 25-60%, has been attributed to multifactorial mechanisms, such as metabolic dysregulation of sorbitol, oxidative stress and neurotrophic deficiency, according to studies on experimental diabetes in rodents and human nerve pathology (Zochodne, 1999; Brownlee, 2001; Kennedy and Zochodne, 2005; Edwards et al, 2008; Bakovic et al, 2018)
Endoneurial capillary morphometry, thrombosis and inflammatory cell infiltration To explore the pattern of microangiopathy, we analyzed the vascular morphometry of diabetic nerves
cluster of differentiation 40 (CD40)(+) cells were significantly increased in the endoneurium of diabetic nerves (63.6±29.4 versus 8.9±8.2 cells/ mm2, P=0.002) and were correlated with the proportion of fibrin(+) vasculatures of diabetic nerves (r=0.73, P=0.0028; Fig. 2L). These results suggested an association of microvascular insults with chronic inflammation, and CD40 might serve as a mediator of enhanced thrombus formation between inflammatory cells and endothelial cells in the diabetic nerves
Summary
Peripheral nerve degeneration in diabetic neuropathy, a major complication of diabetes with a frequency of 25-60%, has been attributed to multifactorial mechanisms, such as metabolic dysregulation of sorbitol, oxidative stress and neurotrophic deficiency, according to studies on experimental diabetes in rodents and human nerve pathology (Zochodne, 1999; Brownlee, 2001; Kennedy and Zochodne, 2005; Edwards et al, 2008; Bakovic et al, 2018). Microangiopathy is listed as one of the major microvascular complications of diabetes, including reduced endoneurial capillary density and endothelial dysfunction, the cascades leading to this cellular pathology and its molecular consequences in diabetic nerves have not yet been systemically investigated. Inflammatory mediators can augment the expression of tissue factor genes in endothelial cells, leading to thrombosis, which, in turn, has been postulated as a cause of leukocyte adhesion and inflammatory response amplification (Libby and Simon, 2001). The CD40 ligand binds to its receptor CD40 and induces tissue factor expression in macrophages and endothelial cells. The relationship between the CD40CD40 ligand system and microangiopathy in diabetic nerves has remained unexplored
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