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Biomedicine & Pharmacotherapy =... Oct 2023Cannabinoids are vasoactive substances that act as key regulators of arterial tone in the blood vessels supplying peripheral tissues and the central nervous system. This...
BACKGROUND AND PURPOSE
Cannabinoids are vasoactive substances that act as key regulators of arterial tone in the blood vessels supplying peripheral tissues and the central nervous system. This study aimed to investigate the potential of R-(+)-WIN55212-2 (WIN), a cannabinoid receptor 1 agonist (CB1), as a treatment for retinal ischemia/reperfusion (I/R) injury.
EXPERIMENTAL APPROACH
Male Wistar rats were subjected to retinal I/R injury by increasing intraocular pressure in the anterior chamber. The rats were randomly divided into four groups: normal control, I/R, vehicle (pre-treated with dimethyl sulfoxide [DMSO] via intraperitoneal injection), and experimental (pre-treated with WIN at a dose of 1 ml/kg via intraperitoneal injection). The rats were sacrificed at different time points of reperfusion (1 hour, 3 hours, 6 hours, and 1 day) after inducing retinal I/R injury, and their retinas were collected for analysis. Oxygen-glucose deprived/reperfusion (OGD/R) was performed by initially perfusing the retinas with oxygenated artificial cerebrospinal fluid (ACSF), then switching to an OGD solution to simulate ischemia, followed by another perfusion with ACSF. Pericyte contraction and the "no-reflow" phenomenon were observed using infrared differential interference contrast (IR-DIC) microscopy and immunohistochemistry. Western blot, enzyme-linked immunosorbent assay (ELISA), and nitric oxide (NO) detection were used to explore the potential mechanism.
KEY RESULTS
In both the OGD/R and I/R models, retinal pericytes exhibited persistent contraction even after reperfusion. The ability of WIN to regulate the tone of retinal pericytes and capillaries was specifically blocked by the BKCa inhibitor iberiotoxin (100 nM). WIN demonstrated a protective effect against retinal I/R injury by preserving blood flow in vessels containing pericytes. Pretreatment with WIN alleviated the persistent contraction and apoptosis of retinal pericytes in I/R-induced rats, accompanied by a reduction in intracellular calcium ion (Ca) concentration. The expression of CB1 decreased in a time-dependent manner in the I/R group. After I/R injury, endothelium-derived nitric oxide (eNOS) levels were reduced at all time points, which was successfully reversed by WIN therapy except for the 1 day group. Additionally, the downregulation of cyclic guanosine monophosphate (cGMP) and BKCa expression at 3 hours, 6 hours, and 1 day after I/R injury was restored by pretreatment of WIN.
CONCLUSIONS & IMPLICATIONS
WIN exerted its protective effects on retinal I/R injury by inhibiting the contraction and apoptosis of pericytes through the CB1-eNOS-cGMP-BKCa signaling pathway, thus ameliorated the occlusion of retinal capillaries.
Topics: Rats; Male; Animals; Pericytes; Microcirculation; Rats, Wistar; Cannabinoid Receptor Agonists; Benzoxazines; Ischemia; Reperfusion Injury
PubMed: 37572634
DOI: 10.1016/j.biopha.2023.115197 -
Stem Cell Reports Jan 2024While studied extensively in model systems, human gastrulation remains obscure. The scarcity of fetal biological material as well as ethical considerations limit our...
While studied extensively in model systems, human gastrulation remains obscure. The scarcity of fetal biological material as well as ethical considerations limit our understanding of this process. In vitro attachment of natural blastocysts shed light on aspects of the second week of human development in the absence of the morphological manifestation of gastrulation. Stem cell-derived blastocyst models, blastoids, provide the opportunity to reconstitute pre- to post-implantation development in vitro. Here we show that upon in vitro attachment, human blastoids self-organize a BRA population and undergo gastrulation. Single-cell RNA sequencing of these models replicates the transcriptomic signature of the human gastrula. Analysis of developmental timing reveals that in both blastoid models and natural human embryos, the onset of gastrulation as defined by molecular markers, can be traced to timescales equivalent to 12 days post fertilization. In all, natural human embryos and blastoid models self-organize primitive streak and mesoderm derivatives upon in vitro attachment.
Topics: Humans; Gastrulation; Gastrula; Embryonic Development; Blastocyst; Mesoderm
PubMed: 38101401
DOI: 10.1016/j.stemcr.2023.11.005 -
Seminars in Cell & Developmental Biology 2024Since their discovery, the Hox genes, with their incredible power to reprogram the identity of complete body regions, a phenomenon called homeosis, have captured the... (Review)
Review
Since their discovery, the Hox genes, with their incredible power to reprogram the identity of complete body regions, a phenomenon called homeosis, have captured the fascination of many biologists. Recent research has provided new insights into the function of Hox proteins in different germ layers and the mechanisms they employ to control tissue morphogenesis. We focus in this review on the ectoderm and mesoderm to highlight new findings and discuss them with regards to established concepts of Hox target gene regulation. Furthermore, we highlight the molecular mechanisms involved the transcriptional repression of specific groups of Hox target genes, and summarize the role of Hox mediated gene silencing in tissue development. Finally, we reflect on recent findings identifying a large number of tissue-specific Hox interactor partners, which open up new avenues and directions towards a better understanding of Hox function and specificity in different tissues.
PubMed: 36517344
DOI: 10.1016/j.semcdb.2022.11.011 -
Nature Communications Nov 2023Despite improvements in medical and surgical therapies, a significant portion of patients with critical limb ischemia (CLI) are considered as "no option" for...
Despite improvements in medical and surgical therapies, a significant portion of patients with critical limb ischemia (CLI) are considered as "no option" for revascularization. In this work, a nitric oxide (NO)-boosted and activated nanovesicle regeneration kit (n-BANK) is constructed by decorating stem cell-derived nanoscale extracellular vesicles with NO nanocages. Our results demonstrate that n-BANKs could store NO in endothelial cells for subsequent release upon pericyte recruitment for CLI revascularization. Notably, n-BANKs enable endothelial cells to trigger eNOS activation and form tube-like structures. Subsequently, eNOS-derived NO robustly recruits pericytes to invest nascent endothelial cell tubes, giving rise to mature blood vessels. Consequently, n-BANKs confer complete revascularization in female mice following CLI, and thereby achieve limb preservation and restore the motor function. In light of n-BANK evoking pericyte-endothelial interactions to create functional vascular networks, it features promising therapeutic potential in revascularization to reduce CLI-related amputations, which potentially impact regeneration medicine.
Topics: Humans; Female; Mice; Animals; Pericytes; Endothelial Cells; Nitric Oxide; Ischemia; Stem Cells; Neovascularization, Physiologic
PubMed: 37957174
DOI: 10.1038/s41467-023-43153-x -
Methods in Molecular Biology (Clifton,... 2024In zebrafish, hematopoietic stem cells (HSCs) are born in the developing aorta during embryogenesis. From the definitive wave of hematopoiesis onward, blood homeostasis...
In zebrafish, hematopoietic stem cells (HSCs) are born in the developing aorta during embryogenesis. From the definitive wave of hematopoiesis onward, blood homeostasis relies on self-renewal and differentiation of progeny of existing HSCs, or clones, rather than de novo generation. Here, we describe an approach to quantify the number and size of HSC clones at various times throughout the lifespan of the animal using a fluorescent, multicolor labeling strategy. The system is based on combining the multicolor Zebrabow system with an inducible, early lateral plate mesoderm and hematopoietic lineage specific cre driver (draculin (drl)). The cre driver can be temporally controlled and activated in early hematopoiesis to introduce a color barcoding unique to each HSC and subsequently inherited by their daughter cells. Clonal diversity and dominance can be investigated in normal development and blood disease progression, such as blood cancers. This adoptable method allows researchers to obtain quantitative insight into clonality-defining events and their contribution to adult hematopoiesis.
Topics: Animals; Colorimetry; Zebrafish; Aorta; Clone Cells; Hematopoietic Stem Cells
PubMed: 37668919
DOI: 10.1007/978-1-0716-3401-1_18 -
Regenerative Therapy Dec 2023Vertebrates form their skeletal tissues from three distinct origins (the neural crest, paraxial mesoderm, and lateral plate mesoderm) through two distinct modes of...
Vertebrates form their skeletal tissues from three distinct origins (the neural crest, paraxial mesoderm, and lateral plate mesoderm) through two distinct modes of ossification (intramembranous and endochondral ossification). Since the paraxial mesoderm generates both intramembranous and endochondral bones, it is thought to give rise to both osteoprogenitors and osteo-chondroprogenitors. However, it remains unclear what directs the paraxial mesoderm-derived cells toward these different fates in distinct skeletal elements during human skeletal development. To answer this question, we need experimental systems that recapitulate paraxial mesoderm-mediated intramembranous and endochondral ossification processes. In this study, we aimed to develop a human pluripotent stem cell (hPSC)-based system that models the human intramembranous ossification process. We found that spheroid culture of the hPSC-derived paraxial mesoderm derivatives generates osteoprogenitors or osteo-chondroprogenitors depending on stimuli. The former induced intramembranous ossification, and the latter endochondral ossification, in mouse renal capsules. Transcriptional profiling supported the notion that bone signatures were enriched in the intramembranous bone-like tissues. Thus, we developed a system that recapitulates intramembranous ossification, and that enables the induction of two distinct modes of ossification by controlling the cell fate of the hPSC-derived paraxial mesoderm derivatives.
PubMed: 37860130
DOI: 10.1016/j.reth.2023.09.017 -
Cells Mar 2024Neuromesodermal progenitors (NMPs), serving as the common origin of neural and paraxial mesodermal development in a large part of the trunk, have recently gained... (Review)
Review
Neuromesodermal progenitors (NMPs), serving as the common origin of neural and paraxial mesodermal development in a large part of the trunk, have recently gained significant attention because of their critical importance in the understanding of embryonic organogenesis and the design of in vitro models of organogenesis. However, the nature of NMPs at many essential points remains only vaguely understood or even incorrectly assumed. Here, we discuss the nature of NMPs, focusing on their dynamic migratory behavior during embryogenesis and the mechanisms underlying their neural vs. mesodermal fate choice. The discussion points include the following: (1) How the sinus rhomboidals is organized; the tissue where the neural or mesodermal fate choice of NMPs occurs. (2) NMPs originating from the broad posterior epiblast are associated with N1 enhancer activity. (3) Tbx6-dependent repression occurs during NMP-derived paraxial mesoderm development. (4) The nephric mesenchyme, a component of the intermediate mesoderm, was newly identified as an NMP derivative. (5) The transition of embryonic tissue development from tissue-specific progenitors in the anterior part to that from NMPs occurs at the forelimb bud axial level. (6) The coexpression of Sox2 and Bra in NMPs is conditional and is not a hallmark of NMPs. (7) The ability of the NMP pool to sustain axial embryo growth depends on Wnt3a signaling in the NMP population. Current in vitro models of NMPs are also critically reviewed.
Topics: Animals; Neural Stem Cells; Mesoderm; Germ Layers; Signal Transduction; Nervous System
PubMed: 38534393
DOI: 10.3390/cells13060549 -
Scientific Reports Aug 2023The damaging effects of sleep deprivation (SD) on brain parenchyma have been extensively studied. However, the specific influence of SD on brain pericytes, a primary...
The damaging effects of sleep deprivation (SD) on brain parenchyma have been extensively studied. However, the specific influence of SD on brain pericytes, a primary component of the blood-brain barrier (BBB) and the neurovascular unit (NVU), is still unclear. The present study examined how acute or repeated SD impairs brain pericytes by measuring the cerebrospinal fluid (CSF) levels of soluble platelet-derived growth factor receptor beta (sPDGFRβ) and quantifying pericyte density in the cortex, hippocampus, and subcortical area of the PDGFRβ-P2A-CreER/tdTomato mice, which predominantly express the reporter tdTomato in vascular pericytes. Our results showed that a one-time 4 h SD did not significantly change the CSF sPDGFRβ level. In contrast, repeated SD (4 h/day for 10 consecutive days) significantly elevated the CSF sPDGFRβ level, implying explicit pericyte damages due to repeated SD. Furthermore, repeated SD significantly decreased the pericyte densities in the cortex and hippocampus, though the pericyte apoptosis status remained unchanged as measured with Annexin V-affinity assay and active Caspase-3 staining. These results suggest that repeated SD causes brain pericyte damage and loss via non-apoptosis pathways. These changes to pericytes may contribute to SD-induced BBB and NVU dysfunctions. The reversibility of this process implies that sleep improvement may have a protective effect on brain pericytes.
Topics: Animals; Mice; Pericytes; Brain; Sleep Deprivation; Receptor, Platelet-Derived Growth Factor beta; Brain Injuries; Mice, Transgenic
PubMed: 37550395
DOI: 10.1038/s41598-023-40138-0 -
Molecular Medicine (Cambridge, Mass.) Feb 2024Pericytes are a vital component of the blood-brain barrier, and their involvement in acute inflammation was recently suggested. However, it remains unclear whether...
BACKGROUND
Pericytes are a vital component of the blood-brain barrier, and their involvement in acute inflammation was recently suggested. However, it remains unclear whether pericytes contribute to hypothalamic chronic inflammation and energy metabolism in obesity. The present study investigated the impact of pericytes on the pathophysiology of obesity by focusing on platelet-derived growth factor (PDGF) signaling, which regulates pericyte functions.
METHODS
Tamoxifen-inducible systemic conditional PDGF receptor β knockout mice (Pdgfrb-KO) and Calcium/calmodulin-dependent protein kinase type IIa (CaMKIIa)-positive neuron-specific PDGF receptor β knockout mice (Pdgfrb-KO) were fed a high-fat diet, and metabolic phenotypes before and 3 to 4 weeks after dietary loading were examined. Intracellular energy metabolism and relevant signal transduction in lipopolysaccharide- and/or platelet-derived growth factor-BB (PDGF-BB)-stimulated human brain pericytes (HBPCs) were assessed by the Seahorse XFe24 Analyzer and Western blotting. The pericyte secretome in conditioned medium from HBPCs was studied using cytokine array kit, and its impact on polarization was examined in bone marrow-derived macrophages (BMDMs), which are microglia-like cells.
RESULTS
Energy consumption increased and body weight gain decreased after high-fat diet loading in Pdgfrb-KO mice. Cellular oncogene fos (cFos) expression increased in proopiomelanocortin (POMC) neurons, whereas microglial numbers and inflammatory gene expression decreased in the hypothalamus of Pdgfrb-KO mice. No significant changes were observed in Pdgfrb-KO mice. In HBPCs, a co-stimulation with lipopolysaccharide and PDGF-BB shifted intracellular metabolism towards glycolysis, activated mitogen-activated protein kinase (MAPK), and modulated the secretome to the inflammatory phenotype. Consequently, the secretome showed an increase in various proinflammatory chemokines and growth factors including Epithelial-derived neutrophil-activating peptide 78 (C-X-C motif chemokine ligand (CXCL)5), Thymus and activation-regulated chemokine (C-C motif chemokine (CCL)17), Monocyte chemoattractant protein 1 (CCL2), and Growth-regulated oncogene α (CXCL1). Furthermore, conditioned medium from HBPCs stimulated the inflammatory priming of BMDMs, and this change was abolished by the C-X-C motif chemokine receptor (CXCR) inhibitor. Consistently, mRNA expression of CXCL5 was elevated by lipopolysaccharide and PDGF-BB treatment in HBPCs, and the expression was significantly lower in the hypothalamus of Pdgfrb-KO mice than in control Pdgfrb mice (FL) following 4 weeks of HFD feeding.
CONCLUSIONS
PDGF receptor β signaling in hypothalamic pericytes promotes polarization of macrophages by changing their secretome and contributes to the progression of obesity.
Topics: Mice; Humans; Animals; Platelet-Derived Growth Factor; Pericytes; Becaplermin; Receptor, Platelet-Derived Growth Factor beta; Culture Media, Conditioned; Lipopolysaccharides; Signal Transduction; Inflammation; Mice, Knockout; Obesity; Hypothalamus; Proto-Oncogene Proteins c-sis
PubMed: 38317079
DOI: 10.1186/s10020-024-00793-z -
A degron-based approach to manipulate Eomes functions in the context of the developing mouse embryo.Proceedings of the National Academy of... Oct 2023The T-box transcription factor Eomesodermin (Eomes), also known as Tbr2, plays essential roles in the early mouse embryo. Loss-of-function mutant embryos arrest at...
The T-box transcription factor Eomesodermin (Eomes), also known as Tbr2, plays essential roles in the early mouse embryo. Loss-of-function mutant embryos arrest at implantation due to Eomes requirements in the trophectoderm cell lineage. Slightly later, expression in the visceral endoderm promotes anterior visceral endoderm formation and anterior-posterior axis specification. Early induction in the epiblast beginning at day 6 is necessary for nascent mesoderm to undergo epithelial to mesenchymal transition (EMT). Eomes acts in a temporally and spatially restricted manner to sequentially specify the yolk sac haemogenic endothelium, cardiac mesoderm, definitive endoderm, and axial mesoderm progenitors during gastrulation. Little is known about the underlying molecular mechanisms governing Eomes actions during the formation of these distinct progenitor cell populations. Here, we introduced a degron-tag and mCherry reporter sequence into the Eomes locus. Our experiments analyzing homozygously tagged embryonic stem cells and embryos demonstrate that the degron-tagged Eomes protein is fully functional. dTAG (degradation fusion tag) treatment in vitro results in rapid protein degradation and recapitulates the Eomes-null phenotype. However in utero administration of dTAG resulted in variable and lineage-specific degradation, likely reflecting diverse cell type-specific Eomes expression dynamics. Finally, we demonstrate that Eomes protein rapidly recovers following dTAG wash-out in vitro. The ability to temporally manipulate Eomes protein expression in combination with cell marking by the mCherry-reporter offers a powerful tool for dissecting Eomes-dependent functional roles in these diverse cell types in the early embryo.
Topics: Mice; Animals; Epithelial-Mesenchymal Transition; T-Box Domain Proteins; Germ Layers; Embryo, Mammalian; Mesoderm; Endoderm; Gene Expression Regulation, Developmental
PubMed: 37871215
DOI: 10.1073/pnas.2311946120