Background Achieving a stable vasculature is crucial for tissue regeneration. Endothelial cells initiate vascular morphogenesis, followed by mural cells that stabilize ne... More
Background Achieving a stable vasculature is crucial for tissue regeneration. Endothelial cells initiate vascular morphogenesis, followed by mural cells that stabilize new vessels. This study investigates the in vivo effects of Sema4D-Plexin-B1 signaling on stem cells from human exfoliated deciduous teeth (SHED)-supported angiogenesis, focusing on its mechanism in PDGF-BB secretion. We also explore macrophages as an endogenous source of Sema4D for vascular stabilization. Methods The in vivo Matrigel plug angiogenesis assay was conducted to examine the impact of Sema4D on vessel formation and stabilization supported by SHED. Knockdown of Plexin-B1 in human umbilical vein endothelial cells (HUVECs) and the application of PDGFR-β inhibitors were utilized to explore the fundamental regulatory mechanisms. Furthermore, the m6A methylation levels of total RNA and the expression of Methyltransferase-like 3 (METTL3) were assessed under conditions of Sema4D treatment in vitro. An ELISA was employed to measure the levels of Sema4D in the supernatants derived from THP-1 cell-mediated macrophages. Additionally, a three-dimensional vasculature-on-a-chip microfluidic device was employed to investigate the role of M2c macrophage-derived Sema4D in the stabilization of vascular structures. Results Sema4D induced the formation of a greater number of perfused vessels by HUVECs and enhanced the coverage of these vessels by SM22α-positive SHED (SM22α+SHED). Conversely, the knockdown of the Plexin-B1 receptor in HUVECs or inhibition of PDGFR-β reversed the Sema4D-induced vascular stabilization, thereby confirming the regulatory role of the Plexin-B1/PDGF-BB axis in the recruitment of mural cells mediated by Sema4D. Mechanistically, Sema4D was found to upregulate the expression of methyltransferases, specifically METTL3, and to elevate the level of m6A modification in HUVECs. This modification was determined to be critical for enhancing PDGF-BB secretion, suggesting that Sema4D activates an epigenetic regulatory mechanism. Additionally, we investigated the secretion of Sema4D by various macrophage phenotypes, identifying that M2c macrophages secrete significant levels of Sema4D. This secretion recruits SM22α+SHED as mural cells by inducing endothelial PDGF production on a vasculature-on-a-chip platform, indicating a potential role for macrophages in facilitating vascular stabilization. Conclusions Sema4D acts on Plexin-B1, inducing METTL3-mediated PDGF-BB secretion to recruit SHED to stabilize vessels. Macrophages could be a key source of Sema4D for vascular stabilization. Less
Traumatic brain injury (TBI) is a major cause of morbidity and mortality worldwide, affecting over 10 million people annually, with an estimated cost of $76.5 billion. Al... More
Traumatic brain injury (TBI) is a major cause of morbidity and mortality worldwide, affecting over 10 million people annually, with an estimated cost of $76.5 billion. Although apocynin freely transverses the blood–brain barrier (BBB), its application is limited due to its rapid elimination, low terminal half-life (t1/2 = 6.7 min), narrow dose–response relationship, and cytotoxicity, thereby requiring repeated dosages. With this study, we aimed to develop transferrin-functionalized nanoparticles encapsulating apocynin to treat neuroinflammation for targeted drug delivery to sites of brain injury. As a preliminary approach, we endeavored to optimize the formulation parameters of apocynin-loaded albumin nanoparticles prepared through the desolvation method. The nanoparticles were characterized for their size, polydispersity, surface charge, drug loading and in vitro drug release. In this study, we also investigated the anti-inflammatory and neuroprotective effects of free apocynin and nanoparticle-loaded apocynin in neuronal cells. We show that the developed formulation displayed monodispersed, nanosized particles with higher entrapment efficiency, loading, stability, and sustained release profiles. The permeability of the nanoparticles across HBMECs reached the maximum at 67%. The in vivo evaluation revealed the enhanced uptake of transferrin-anchored nanoparticles in the brain tissues when compared with unmodified nanoparticles after I.V. administration. In vivo nanoparticle localization studies using a blast TBI (bTBI) model and confocal fluorescence microscopy have shown that tf-apoANPs are successful in delivering relatively high amounts of nanoparticles to the brain parenchyma and glial cells compared to non-targeted nanoparticles. We also establish that targeted nanoparticles accumulate in the brain. In conclusion, tf-apoANPs are efficacious carriers for targeted delivery across the blood–brain barrier to potentially treat neuroinflammation in brain injury and other diseases.
Keywords:
apocynin; nanoparticle; albumin; HPLC; desolvation method; targeted delivery; biodistribution; neuroprotection Less
Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional m... More
Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50–100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair. Less
