Central role of endogenous Toll-like receptor-2 activation in regulating inflammation, reactive oxygen species production, and subsequent neointimal formation after vascular injury

Abstract Background PTEN-induced kinase 1 (PINK1) is a serine/threonine-protein kinase in mitochondria that is critical for mitochondrial quality control. PINK1 triggers mitophagy, a selective autophagy of mitochondria, and is involved in mitochondrial regeneration. Although increments of mitochondrial biogenesis and activity are known to be crucial during differentiation, data regarding the specific role of PINK1 in osteogenic maturation and bone remodeling are limited. Methods We adopted an ovariectomy model in female wildtype and Pink1−/− mice. Ovariectomized mice were analyzed using micro-CT, H&E staining, Masson’s trichrome staining. RT-PCR, western blot, immunofluorescence, alkaline phosphatase, and alizarin red staining were performed to assess the expression of PINK1 and osteogenic markers in silencing of PINK1 MC3T3-E1 cells. Clinical relevance of PINK1 expression levels was determined via qRT-PCR analysis in normal and osteoporosis patients. Results A significant decrease in bone mass and collagen deposition was observed in the femurs of Pink1−/− mice after ovariectomy. Ex vivo, differentiation of osteoblasts was inhibited upon Pink1 downregulation, accompanied by impaired mitochondrial homeostasis, increased mitochondrial reactive oxygen species production, and defects in mitochondrial calcium handling. Furthermore, PINK1 expression was reduced in bones from patients with osteoporosis, which supports the practical role of PINK1 in human bone disease. Conclusions In this study, we demonstrated that activation of PINK1 is a requisite in osteoblasts during differentiation, which is related to mitochondrial quality control and low reactive oxygen species production. Enhancing PINK1 activity might be a possible treatment target in bone diseases as it can promote a healthy pool of functional mitochondria in osteoblasts.

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Toll-Like Receptor 3 Activation Is Involved in Venous Thrombosis Development,

Blood ◽

10.1182/blood.v118.21.3277.3277 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3277-3277

Author(s):

Catherine A Lemarie ◽

Sandrine Laurance ◽

Mark Blostein

Keyword(s):

Reactive Oxygen Species ◽

Endothelial Cells ◽

Venous Thrombosis ◽

Intravenous Injection ◽

Reactive Oxygen Species Production ◽

Reactive Oxygen ◽

Oxygen Species ◽

Toll Like Receptor ◽

Double Stranded Rna ◽

Species Production

Abstract Abstract 3277 Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. Inflammation may have a role in both the genesis and resolution of venous thrombi. Although septic thrombophlebitis occurs, most DVT are sterile. Recently, the role of toll-like receptor (TLR) signaling in modulating sterile inflammation has become better defined in experimental injury models. TLR3, for example, is activated by viral long double stranded RNA and endogenous RNA released by cellular apoptosis and necrosis, and induce a proinflammatory cellular response. Short single-stranded RNA, such as those used in RNA inhibition, stimulate TLR3 in endothelial cells and inhibit neoangiogenesis. Thus, we hypothesize that TLR3 might be involved in the inflammatory development of venous thrombosis. We first investigated whether stimulation of TLR3 influences venous thrombosis in mice. Intravenous injection of polyinosine polycytidylic acid (polyI:C), a synthetic double-stranded RNA analog and TLR3 ligand, increase the size and the cellular density of thrombi after FeCl3-induced inferior vena cava injury. TLR3 stimulation with polyI:C is also associated with an increased reactive oxygen species production, as well as increased neutrophil and macrophage content. Using a specific fluorescent probe for RNA, we found that FeCl3 induces RNA release and thus increases the RNA content in the thrombus. We next investigated the effect of RNase on thrombus development. Intravenous injection of RNase decreased thrombus size, as well as reactive oxygen species production and neutrophil and macrophage content in the thrombus. In vitro incubation of endothelial cells with polyI:C induces production of pro-inflammatory cytokines GM-CSF, G-CSF, IL-6, IL-8 and interferon-γ-induced protein 10 involved in the recruitment of neutrophils and macrophages. Taken together, these results strongly suggest that RNA release and TLR3 stimulation after endothelial cell injury participate in thrombus formation by inducing a pro-inflammatory response leading to the recruitment of neutrophils and macrophages. Disclosures: No relevant conflicts of interest to declare.

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