Biology subjects

Abdelazim, H.

Publications and source records attributed to Abdelazim, H..

4 recordsLinked to original sources

bioRxiv2025

Pericyte and Endothelial Cell Responses within Murine Cerebral Capillaries After Blood Flow Cessation

ABSTRACT/SUMMARYBlood flow provides critical inputs for mechanisms governing vascular homeostasis. Altered hemodynamics can therefore trigger a wide range of cellular responses in blood vessels. Endothelial cells (ECs) downstream of atherosclerotic plaques for instance are exposed to turbulent flow, activating inflammatory pathways that promote immune cell infiltration. In conditions like stroke and myocardial infarction, the abrupt loss of blood flow prompts responses in vascular cells such as ECs and pericytes (PCs) to adapt to ischemic or no-flow conditions. To better understand how cerebral capillary ECs and PCs react to the sudden loss of blood flow, we used a murine brain slice model cultured for 12- and 24-hours in artificial cerebrospinal fluid (aCSF) with 95% oxygen supplementation. As expected, inflammation mediators were upregulated in cultured slices compared to non-cultured samples, particularly those associated with leukocyte recruitment. Additionally, transcriptional markers of extracellular matrix (ECM) remodeling and cell-ECM interactions were elevated, consistent with reduced PC coverage along capillaries. We initially presumed these changes reflected blood-brain barrier (BBB) degradation, but instead we found an increase in mRNA transcripts for EC junctions and stable protein levels for junction molecules, with an apparent rearrangement of Claudin5-based tight junctions. Some capillaries also exhibited reduced diameters, suggesting constriction by PCs or a subset thereof. Consistent with these observations, we found an upregulation of the vasoconstrictor Endothelin-1 (ET-1) with its receptors and contractile proteins found in a subpopulation of PCs. Suppressing ET-1 activity prevented Claudin5 upregulation, indicating that ET-1 might regulate microvascular constriction and associated changes in endothelial tight junctions. Overall, these results suggest that in the absence of blood flow, PCs contribute to capillary wall remodeling by (i) potentially mediating a mechanism driven by ET-1 that affects EC Claudin5 dynamics, and (ii) reducing capillary ECM and detaching from microvessel walls.

cell biology↗

bioRxiv2024

Optimized Enrichment of Murine Blood-Brain Barrier Vessels with a Critical Focus on Network Hierarchy in Post-Collection Analysis

Cerebrovascular networks contain a unique region of interconnected capillaries known as the blood-brain barrier (BBB). Positioned between upstream arteries and downstream veins, these microvessels have unique structural features, such as the absence of vascular smooth muscle cells (vSMCs) and a relatively thin basement membrane, to facilitate highly efficient yet selective exchange between the circulation and the brain interstitium. This vital role in neurological health and function has garnered significant attention from the scientific community and inspired methodology for enriching BBB capillaries. Extensive characterization of the isolates from such protocols is essential for framing the results of follow-on experiments and analyses, providing the most accurate interpretation and assignment of BBB properties. Seeking to aid in these efforts, here we visually screened output samples using fluorescent labels and found considerable reduction of non-vascular cells following density gradient centrifugation (DGC) and subsequent filtration. Comparatively, this protocol enriched brain capillaries, though larger diameter vessels associated with vSMCs could not be fully excluded. Protein analysis further underscored the enrichment of vascular markers following DGC, with filtration preserving BBB-associated markers and reducing - though not fully removing - arterial/venous contributions. Transcriptional profiling followed similar trends of DGC plus filtration generating isolates with less non-vascular and non- capillary material included. Considering vascular network hierarchy inspired a more comprehensive assessment of the material yielded from brain microvasculature isolation protocols. This approach is important for providing an accurate representation of the cerebrovascular segments being used for data collection and assigning BBB properties specifically to capillaries relative to other regions of the brain vasculature. HIGHLIGHTS- We optimized a protocol for the enrichment of murine capillaries using density gradient centrifugation and follow-on filtration. - We offer an approach to analyzing post-collection cerebrovascular fragments and cells with respect to vascular network hierarchy. - Assessing arterial and venous markers alongside those associated with the BBB provides a more comprehensive view of material collected. - Enhanced insight into isolate composition is critical for a more accurate view of BBB biology relative to larger diameter cerebrovasculature. MOTIVATIONThe recent surge in studies investigating the cerebrovasculature, and the blood-brain barrier (BBB) in particular, has inspired a broad range of approaches to target and observe these specialized blood vessels within murine models. To capture transcriptional and molecular changes during a specific intervention or disease model, techniques have been developed to isolate brain capillary networks and collect their cellular constituents for downstream analysis. Here, we sought to highlight the benefits and cautions of isolating and enriching microvessels from murine brain tissue. Specifically, through rigorous assessment of the output material following application of specific protocols, we presented the benefits of specific approaches to reducing the inclusion of non-vascular cells and non-capillary vessel segments, verified by analysis of vascular-related proteins and transcripts. We also emphasized the levels of larger- caliber vessels (i.e. arteries/arterioles and veins/venules) that are collected alongside cerebral capillaries with each method. Distinguishing these vascular regions with greater precision is critical for attributing specific characteristics exclusively to the BBB where metabolic, ion, and waste exchange occurs. While the addition of larger vessels to molecular / transcriptional analyses or follow-on experiments may not be substantial for a given protocol, it is essential to gauge and report their level of inclusion, as their contributions may be inadvertently assigned to the BBB. Therefore, we present this optimized brain microvessel isolation protocol and associated evaluation methods to underscore the need for increased rigor in characterizing vascular regions that are collected and analyzed within a given study. Graphical AbstractConstructed using resources from BioRender.com. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/613898v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@7b4448org.highwire.dtl.DTLVardef@bff92borg.highwire.dtl.DTLVardef@17fa8b3org.highwire.dtl.DTLVardef@1c950d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

A Soluble Platelet-Derived Growth Factor Receptor-β Originates via Pre-mRNA Splicing in the Healthy Brain and is Differentially Regulated during Hypoxia and Aging

ABSTRACT/SUMMARYThe platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFR{beta}) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases (RTKs) like PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFR{beta} (sPDGFR{beta}) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFR{beta} variants, and specifically during tissue homeostasis. Here, we found sPDGFR{beta} protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFR{beta} isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFR{beta} by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFR{beta} transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFR{beta} protein was detected throughout the brain parenchyma in distinct regions such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFR{beta} variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFR{beta} variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFR{beta} likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFR{beta} in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion - critical processes underlying neuronal health and function, and in turn memory and cognition.

cell biology↗

bioRxiv2022

Pericyte Heterogeneity Identified by 3D Ultrastructural Analysis of the Microvessel Wall

ABSTRACT/SUMMARYUnequivocal pericyte identification remains a limitation in the field of vascular biology given the lack of unique molecular marker. Compounding this challenge are the recently described heterogeneities in pericyte morphology across microvascular networks. Here, we found further support on the ultrastructural level for classifying pericytes into sub-types - "thin-strand" (TSPs), mesh (MP), and ensheathing (EP) pericytes - based on their architecture in the mouse brain microcirculation. We also observed several instances of an additional cell type in the medial layer between endothelial cells and pericytes, specifically associated with EPs. A conserved characteristic across PC subtypes was extracellular matrix (ECM) encompassing the vascular unit and dispersed among neighboring cells. ECM thicknesses fell within a specific range depending on vessel location, and only thinned where cells were in closer proximity. Pericytes and endothelial cells formed "peg-and-socket" structures at these locations, providing another distinguishing feature across PC subtypes. Unique contact locations seemed to be present between medial and endothelial cells, as well as between vascular cells and the brain parenchyma. The ECM surrounding EPs exhibited another notable configuration in that thin extensions radiated out from the vessel wall into the surrounding parenchyma, suggesting mechanical and/or biochemical roles. Considering these data together, ultrastructural observations may provide an orthogonal perspective on pericyte heterogeneity and the presence of medial cells in cerebrovascular walls as well as assessing ECM coverage as a criterion for PC identification and exploring PC-associated ECM extensions that may have unique relevance in health and disease.

cell biology↗