Hypoxia-ischaemia (HI) can induce the death of cerebrovascular constituent cells through oxidative stress. Hydrogen is a powerful antioxidant which can activate the antioxidant system. A hypoxia-ischaemia brain damage (HIBD) model was established in 7-day-old SD rats. Rats were treated with different doses of hydrogen-rich water (HRW), and brain pericyte oxidative stress damage, cerebrovascular function and brain tissue damage were assessed. Meanwhile, in vitro-cultured pericytes were subjected to oxygen-glucose deprivation and treated with different concentrations of HRW. Oxidative injury was measured and the molecular mechanism of how HRW alleviated oxidative injury of pericytes was also examined. The results showed that HRW significantly attenuated HI-induced oxidative stress in the brain pericytes of neonatal rats, partly through the Nrf2-HO-1 pathway, further improving cerebrovascular function and reducing brain injury and dysfunction. Furthermore, HRW is superior to a single-cell death inhibitor for apoptosis, ferroptosis, parthanatos, necroptosis and autophagy and can better inhibit HI-induced pericyte death. The liver and kidney functions of rats were not affected by present used HRW dose. This study elucidates the role and mechanism of hydrogen in treating HIBD from the perspective of pericytes, providing new theoretical evidence and mechanistic references for the clinical application of hydrogen in neonatal HIE.

Pericytes are a distinct type of cells interacting with endothelial cells in blood vessels and contributing to endothelial barrier integrity. Furthermore, pericytes show mesenchymal stem cell properties. Muscle-derived pericytes can demonstrate both angiogenic and myogenic capabilities. It is well known that regenerative abilities and muscle stem cell potential decline during aging, leading to sarcopenia. Therefore, this study aimed to investigate the potential of pericytes in supporting muscle differentiation and angiogenesis in elderly individuals and in patients affected by Ullrich congenital muscular dystrophy or by Bethlem myopathy, two inherited conditions caused by mutations in collagen VI genes and sharing similarities with the progressive skeletal muscle changes observed during aging. The study characterized pericytes from different age groups and from individuals with collagen VI deficiency by mass spectrometry-based proteomic and bioinformatic analyses. The findings revealed that aged pericytes display metabolic changes comparable to those seen in aging skeletal muscle, as well as a decline in their stem potential, reduced protein synthesis, and alterations in focal adhesion and contractility, pointing to a decrease in their ability to form blood vessels. Strikingly, pericytes from young patients with collagen VI deficiency showed similar characteristics to aged pericytes, but were found to still handle oxidative stress effectively together with an enhanced angiogenic capacity.

Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) and the failure of axonal growth. SCI activates a complex series of responses, including cell apoptosis and endoplasmic reticulum (ER) stress. Pericytes play a critical role in maintaining BSCB integrity and facilitating tissue growth and repair. However, the roles of pericytes in SCI and the potential mechanisms underlying the improvements in functional recovery in SCI remain unclear. Recent evidence indicates that irisflorentin exerts neuroprotective effects against Parkinson\'s disease; however, whether it has potential protective roles in SCI or not is still unknown. In this study, we found that the administration of irisflorentin significantly inhibited pericyte apoptosis, protected BSCB integrity, promoted axonal growth, and ultimately improved locomotion recovery in a rat model of SCI. In vitro, we found that the positive effects of irisflorentin on axonal growth were likely to be mediated by regulating the crosstalk between pericytes and neurons. Furthermore, irisflorentin effectively ameliorated ER stress caused by incubation with thapsigargin (TG) in pericytes. Meanwhile, the protective effect of irisflorentin on BSCB disruption is strongly related to the reduction of pericyte apoptosis via inhibition of ER stress. Collectively, our findings demonstrate that irisflorentin is beneficial for functional recovery after SCI and that pericytes are a valid target of interest for future SCI therapies.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with neurological sequelae including haemorrhage, thrombosis and ischaemic necrosis and encephalitis. However, the mechanism by which this occurs is unclear. Neurological disease associated with COVID-19 has been proposed to occur following direct infection of the central nervous system and/or indirectly by local or systemic immune activation. We evaluated the expression of angiotensin-converting enzyme-2 and transmembrane protease, serine 2 (TMPRSS2) in brain tissue from five healthy human donors and observed low-level expression of these proteins in cells morphologically consistent with astrocytes, neurons and choroidal ependymal cells within the frontal cortex and medulla oblongata. Primary human astrocytes, neurons, choroid plexus epithelial cells and pericytes supported productive SARS-CoV-2 infection with ancestral, Alpha, Delta and Omicron variants. Infected cells supported the full viral life cycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 can infect human brain cells, and the mechanism of viral entry warrants further investigation.

BACKGROUND: There is significant interest in isolating cells of the blood-brain barrier (BBB) for use in in vitro screening of therapeutics and analyzing cell specific roles in neurovascular pathology. Primary brain cells play an advantageous role in BBB models; however, isolation procedures often do not produce cells at high enough yields for experiments. In addition, although numerous reports provide primary cell isolation methods, the field is lacking in documentation and detail of expected morphological changes that occur throughout culturing and there are minimal troubleshooting resources. Here, we present simplified, robust, and reproducible methodology for isolating astrocytes, pericytes, and endothelial cells, and demonstrate several morphological benchmarks for each cell type throughout the process and culture timeframe. We also analyze common considerations for developing neurovascular cell isolation procedures and recommend solutions for troubleshooting.
RESULTS: The presented methodology isolated astrocytes, pericytes, and endothelial cells and enabled cell attachment, maturation, and cell viability. We characterized milestones in cell maturation over 12 days in culture, a common timeline for applications of these cell types in BBB models. Phase contrast microscopy was used to show initial cell plating, attachment, and daily growth of isolated cells. Confocal microscopy images were analyzed to determine the identity of cell types and changes to cell morphology. Nuclear staining was also used to show the viability and proliferation of glial cells at four time points. Astrocyte branches became numerous and complex with increased culture time. Microglia, oligodendrocytes, and neurons were present in mixed glial cultures for 12 days, though the percentage of microglia and neurons expectedly decreased after passaging, with microglia demonstrating a less branched morphology.
CONCLUSIONS: Neurovascular cells can be isolated through our optimized protocols that minimize cell loss and encourage the adhesion and proliferation of isolated cells. By identifying timepoints of viable glia and neurons within an astrocyte-dominant mixed culture, these cells can be used to evaluate drug targeting, uptake studies, and response to pathological stimulus in the neurovascular unit.

PDGF receptors play pivotal roles in both developmental and physiological processes through the regulation of mesenchymal cells involved in paracrine instructive interactions with epithelial or endothelial cells. Tumor biology studies, alongside analyses of patient tissue samples, provide strong indications that the PDGF signaling pathways are also critical in various types of human cancer. This review summarizes experimental findings and correlative studies, which have explored the biological mechanisms and clinical relevance of PDGFRs in mesenchymal cells of the tumor microenvironment. Collectively, these studies support the overall concept that the PDGF system is a critical regulator of tumor growth, metastasis, and drug efficacy, suggesting yet unexploited targeting opportunities. The inter-patient variability in stromal PDGFR expression, as being linked to prognosis and treatment responses, not only indicates the need for stratified approaches in upcoming therapeutic investigations but also implies the potential for the development of PDGFRs as biomarkers of clinical utility, interestingly also in settings outside PDGFR-directed treatments.

Preterm infants can face lasting neurodevelopmental challenges due to hypoxia-induced injury of the cerebral white matter. In this issue of Neuron, Ren et al.1 identify microvascular pericytes as unexpected targets for growth hormone signaling, which enhances angiogenesis and remyelination after hypoxic injury in the developing mouse brain.

Mural cells are critically important for the development, maturation, and maintenance of the blood vasculature. Pericytes are predominantly observed in capillaries and venules, while vascular smooth muscle cells (VSMCs) are found in arterioles, arteries, and veins. In this study, we have investigated functional differences between human pericytes and human coronary artery smooth muscle cells (CASMCs) as a model VSMC type. We compared the ability of these two mural cells to invade three-dimensional (3D) collagen matrices, recruit to developing human endothelial cell (EC)-lined tubes in 3D matrices and induce vascular basement membrane matrix assembly around these tubes. Here, we show that pericytes selectively invade, recruit, and induce basement membrane deposition on EC tubes under defined conditions, while CASMCs fail to respond equivalently. Pericytes dramatically invade 3D collagen matrices in response to the EC-derived factors, platelet-derived growth factor (PDGF)-BB, PDGF-DD, and endothelin-1, while minimal invasion occurs with CASMCs. Furthermore, pericytes recruit to EC tube networks, and induce basement membrane deposition around assembling EC tubes (narrow and elongated tubes) when these cells are co-cultured. In contrast, CASMCs are markedly less able to perform these functions showing minimal recruitment, little to no basement membrane deposition, with wider and shorter tubes. Our new findings suggest that pericytes demonstrate much greater functional ability to invade 3D matrix environments, recruit to EC-lined tubes and induce vascular basement membrane matrix deposition in response to and in conjunction with ECs.

Mesenchymal cells within theplacental villi play a crucial role in shaping the morphology of branching structures and driving the development of blood vessels. However, the markers and functions of placental villous pericytes (PVPs) as distinct subgroups of placental villous mesenchymal cells, remain unclear. Therefore, in this study, the markers and functions of PVPs were investigated. Single-cell sequencing data from the first-trimester placental villi was obtained and the Seurat tool was used to identify PVP markers. Gene Ontology (GO) analysis of specific genes was performed using the DAVID database. The Cellchat tool was employed to investigate the interaction signals between the PVPs and other cells. Expression of the PVP markers was confirmed using immunofluorescence. Presence of extracellular vesicles in the placental villous mesenchyme and PVPs was examined by transmission electron microscopy. Our findings revealed that renin (REN) and amphiregulin (AREG)-positive fibroblasts in the placental villi specifically expressed several classic pericyte markers. In the first trimester, certain conserved functions of pericytes were observed and they displayed tissue-specific functions such as in the integrin-mediated signaling pathway and extracellular exosomes. Moreover, the placental villous mesenchyme was found to be rich in extracellular vesicles. AREG is specifically transcribed in the first trimester PVPs, however, its protein was located in syncytiotrophoblasts. These insights contribute to a comprehensive understanding of early placental development and offer new therapeutic targets for placenta-derived pregnancy complications.

Although the concept that the blood-brain barrier (BBB) plays an important role in the etiology and pathogenesis of Alzheimer\'s disease (AD) has become increasingly accepted, little is known yet about how it actually contributes. We and others have recently identified a novel functionally distinct subset of BBB pericytes (PCs). In the present study, we sought to determine whether these PC subsets differentially contribute to AD-associated pathologies by immunohistochemistry and amyloid beta (Aβ) peptidomics. We demonstrated that a disease-associated PC subset (PC2) expanded in AD patients compared to age-matched, cognitively unimpaired controls. Surprisingly, we found that this increase in the percentage of PC2 (%PC2) was correlated negatively with BBB breakdown in AD patients, unlike in natural aging or other reported disease conditions. The higher %PC2 in AD patients was also correlated with a lower Aβ42 plaque load and a lower Aβ42:Aβ40 ratio in the brain as determined by immunohistochemistry. Colocalization analysis of multicolor confocal immunofluorescence microscopy images suggests that AD patient with low %PC2 have higher BBB breakdown due to internalization of Aβ42 by the physiologically normal PC subset (PC1) and their concomitant cell death leading to more vessels without PCs and increased plaque load. On the contrary, it appears that PC2 can secrete cathepsin D to cleave and degrade Aβ built up outside of PC2 into more soluble forms, ultimately contributing to less BBB breakdown and reducing Aβ plaque load. Collectively our data shows functionally distinct mechanisms for PC1 and PC2 in high Aβ conditions, demonstrating the importance of correctly identifying these populations when investigating the contribution of neurovascular dysfunction to AD pathogenesis.