Human serum fibrinogen induces brain microvascular endothelial dysfunction and blood-brain barrier dysregulation via dynamin related protein 1 dependent pathway

Background: Fibrinogen (Fgn) is one of several pathogenic factors in the brain microvasculature. It is upregulated in the blood of the aged, Alzheimer’s disease, and other neurological disease patients. We explored the possible role of a human serum Fgn (hFgn) mediated mechanism for endothelial and blood-brain barrier (BBB) dysregulation in primary human brain microvascular endothelial cells (HBMEC). Methods: To assess the endothelial and BBB dysregulation ex vivo, we measured protein expression change by western blot and immunofluorescence in hFgn-treated HBMEC. Cell viability was determined by NAD(P)H-dependent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay, and in vitro BBB-permeability was assessed by transwell migration assay. Stable DRP1 knockdown cells were developed by transduction of shRNA(h) lentivirus particles in HBMEC. Results: Clinical studies have reported that Fgn is an abundant protein in human blood plasma at concentrations ranging from 1.5–4 mg/mL with a normal half-life of 3–5 days, and the plasma concentration of Fgn increased progressively with age in healthy subjects. We observed that 4 mg/mL of hFgn induced significant cytotoxicity and generated mitochondrial reactive oxygen species in HBMEC within 24 h. With the same concentration of Fgn, we observed that most of the mitochondria were elongated or hyperfused, possibly due to significant reduction of phosphorylated dynamin related protein 1 (pDRP1[S616]) in hFgn-treated HBMEC. Moreover, mitochondrial oxidative phosphorylation (OXPHOS)-related mitochondrial complex-II, -III, and -V proteins were also decreased in hFgn-treated HBMEC. Interestingly, autophagy-related, microtubule-associated protein light chain 3B-II (LC3B-II) and lysosomal membrane-associated protein 2 (LAMP2) decreased, while a negative regulator of autophagy, p62, increased in hFgn-treated HBMEC, suggesting hFgn-mediated induction of defective autophagy. Importantly, hFgn induced BBB permeability, possibly due to loss of several BBB-related proteins such as JAM-A, occludin, ZO-1/-2, and PECAM-1 in HBMEC. DRP1 knockdown by shRNA(h) resulted in reduction of OXPHOS and BBB-related proteins as well as defective autophagy induction in HBMEC. Conclusions: Our results suggest that detrimental effects of elevated Fgn downregulates DRP1, leading to endothelial and BBB dysfunction in the brain microvasculature, which affects neuronal health/function and adds vulnerability to strokes and neurological diseases. HL148836, AG063345, NS114286, AG047296, and AG074489. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.