A Soluble Platelet-Derived Growth Factor Receptor-ß Originates via Pre-mRNA Splicing in the Healthy Brain and Is Upregulated during Hypoxia and Aging.

The 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 (PDGFRß) 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 such as 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 PDGFRß (sPDGFRß) 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 sPDGFRß variants, and specifically during tissue homeostasis. Here, we found sPDGFRß 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 sPDGFRß 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 sPDGFRß by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRß transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRß 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 sPDGFRß variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRß variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRß 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 sPDGFRß 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.

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

The 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 (PDGFRß) 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 PDGFRß (sPDGFRß) 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 sPDGFRß variants, and specifically during tissue homeostasis. Here, we found sPDGFRß 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 sPDGFRß 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 sPDGFRß by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRß transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRß 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 sPDGFRß variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRß variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRß 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 sPDGFRß 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.

Pericyte Progenitor Coupling to the Emerging Endothelium During Vasculogenesis via Connexin 43.

BACKGROUND: Vascular pericytes stabilize blood vessels and contribute to their maturation, while playing other key roles in microvascular function. Nevertheless, relatively little is known about involvement of their precursors in the earliest stages of vascular development, specifically during vasculogenesis. METHODS: We combined high-power, time-lapse imaging with transcriptional profiling of emerging pericytes and endothelial cells in reporter mouse and cell lines. We also analyzed conditional transgenic animals deficient in Cx43/Gja1 (connexin 43/gap junction alpha-1) expression within Ng2+ cells. RESULTS: A subset of Ng2-DsRed+ cells, likely pericyte/mural cell precursors, arose alongside endothelial cell differentiation and organization and physically engaged vasculogenic endothelium in vivo and in vitro. We found no overlap between this population of differentiating pericyte/mural progenitors and other lineages including hemangiogenic and neuronal/glial cell types. We also observed cell-cell coupling and identified Cx43-based gap junctions contributing to pericyte-endothelial cell precursor communication during vascular assembly. Genetic loss of Cx43/Gja1 in Ng2+ pericyte progenitors compromised embryonic blood vessel formation in a subset of animals, while surviving mutants displayed little-to-no vessel abnormalities, suggesting a resilience to Cx43/Gja1 loss in Ng2+ cells or potential compensation by additional connexin isoforms. CONCLUSIONS: Together, our data suggest that a distinct pericyte lineage emerges alongside vasculogenesis and directly communicates with the nascent endothelium via Cx43 during early vessel formation. Cx43/Gja1 loss in pericyte/mural cell progenitors can induce embryonic vessel dysmorphogenesis, but alternate connexin isoforms may be able to compensate. These data provide insight that may reshape the current framework of vascular development and may also inform tissue revascularization/vascularization strategies.

A Comparison of PET Tracers in Recurrent High-Grade Gliomas: A Systematic Review.

Humans with high-grade gliomas have a poor prognosis, with a mean survival time of just 12-18 months for patients who undergo standard-of-care tumor resection and adjuvant therapy. Currently, surgery and chemoradiotherapy serve as standard treatments for this condition, yet these can be complicated by the tumor location, growth rate and recurrence. Currently, gadolinium-based, contrast-enhanced magnetic resonance imaging (CE-MRI) serves as the predominant imaging modality for recurrent high-grade gliomas, but it faces several drawbacks, including its inability to distinguish tumor recurrence from treatment-related changes and its failure to reveal the entirety of tumor burden (de novo or recurrent) due to limitations inherent to gadolinium contrast. As such, alternative imaging modalities that can address these limitations, including positron emission tomography (PET), are worth pursuing. To this end, the identification of PET-based markers for use in imaging of recurrent high-grade gliomas is paramount. This review will highlight several PET radiotracers that have been implemented in clinical practice and provide a comparison between them to assess the efficacy of these tracers.

Neutrophil Count on Admission Predicts Acute Symptomatic Hydrocephalus After Aneurysmal Subarachnoid Hemorrhage.

OBJECTIVE: To investigate the association between immunologic counts on admission and acute symptomatic hydrocephalus after aneurysmal subarachnoid hemorrhage (aSAH). METHODS: We conducted a retrospective analysis of 143 consecutive patients with aSAH. Patient demographics, clinical parameters, laboratory values, and radiographic imaging were obtained. Univariate and multivariate logistic regression analyses were performed to investigate parameters independently associated with acute symptomatic hydrocephalus. Receiver operating characteristic (ROC) curve analysis determined the best threshold value of neutrophil count to differentiate patients with and without hydrocephalus. RESULTS: Overall, acute symptomatic hydrocephalus developed in 39.16% of patients. In an adjusted multivariate logistic regression model, Hunt and Hess grade 4-5 (odds ratio [OR]: 16.052, 95% confidence interval [CI]: 1.188-216.983; P = 0.037), modified Fisher score 3-4 (OR: 10.107, 95% CI: 1.715-59.572; P = 0.011), intraventricular hemorrhage (OR: 4.578, 95% CI: 1.417-14.788; P = 0.011), neutrophil count (OR: 1.183, 95% CI: 1.033-1.354; P = 0.015), and prior ischemic stroke (OR: 7.003, 95% CI: 1.293-37.929; P = 0.024) were significantly associated with hydrocephalus. ROC analysis for neutrophil count confirmed an acceptable area under the curve (AUC 0.780, 95% CI: 0.701-0.859; P < 0.001). The best threshold value of neutrophil count to predict hydrocephalus was ≥9.80 × 103/mL. Overall, 81.25% of patients who developed shunt dependence had a neutrophil count ≥9.80 × 103/mL on admission (P = 0.003). CONCLUSIONS: Neutrophil count ≥9.80 × 103/mL on admission predicts acute symptomatic hydrocephalus after aSAH in an adjusted multivariate logistic regression model. Moreover, shunt dependence was associated with higher neutrophil counts.

Pericyte migration and proliferation are tightly synchronized to endothelial cell sprouting dynamics.

Pericytes are critical for microvascular stability and maintenance, among other important physiological functions, yet their involvement in vessel formation processes remains poorly understood. To gain insight into pericyte behaviors during vascular remodeling, we developed two complementary tissue explant models utilizing 'double reporter' animals with fluorescently-labeled pericytes and endothelial cells (via Ng2:DsRed and Flk-1:eGFP genes, respectively). Time-lapse confocal imaging of active vessel remodeling within adult connective tissues and embryonic skin revealed a subset of pericytes detaching and migrating away from the vessel wall. Vessel-associated pericytes displayed rapid filopodial sampling near sprouting endothelial cells that emerged from parent vessels to form nascent branches. Pericytes near angiogenic sprouts were also more migratory, initiating persistent and directional movement along newly forming vessels. Pericyte cell divisions coincided more frequently with elongating endothelial sprouts, rather than sprout initiation sites, an observation confirmed with in vivo data from the developing mouse brain. Taken together, these data suggest that (i) pericyte detachment from the vessel wall may represent an important physiological process to enhance endothelial cell plasticity during vascular remodeling, and (ii) pericyte migration and proliferation are highly synchronized with endothelial cell behaviors during the coordinated expansion of a vascular network.

Pericytes in Vascular Development.

PURPOSE OF REVIEW: Pericytes are essential components of capillaries in many tissues and organs, contributing to vessel stability and integrity, with additional contributions to microvascular function still being discovered. We review current and foundational studies identifying pericyte differentiation mechanics and their roles in the earliest stages of vessel formation. RECENT FINDINGS: Recent advances in pericyte-focused tools and models have illuminated critical aspects of pericyte biology including their roles in vascular development.Pericytes likely collaborate with endothelial cells undergoing vasculogenesis, initiating direct interactions during sprouting and intussusceptive angiogenesis. Pericytes also provide important regulation of vascular growth including mechanisms underlying vessel pruning, rarefaction, and subsequent regrowth.

Blood Vessel Patterning on Retinal Astrocytes Requires Endothelial Flt-1 (VEGFR-1).

Feedback mechanisms are critical components of many pro-angiogenic signaling pathways that keep vessel growth within a functional range. The Vascular Endothelial Growth Factor-A (VEGF-A) pathway utilizes the decoy VEGF-A receptor Flt-1 to provide negative feedback regulation of VEGF-A signaling. In this study, we investigated how the genetic loss of flt-1 differentially affects the branching complexity of vascular networks in tissues despite similar effects on endothelial sprouting. We selectively ablated flt-1 in the post-natal retina and found that maximum induction of flt-1 loss resulted in alterations in endothelial sprouting and filopodial extension, ultimately yielding hyper-branched networks in the absence of changes in retinal astrocyte architecture. The mosaic deletion of flt-1 revealed that sprouting endothelial cells flanked by flt-1-/- regions of vasculature more extensively associated with underlying astrocytes and exhibited aberrant sprouting, independent of the tip cell genotype. Overall, our data support a model in which tissue patterning features, such as retinal astrocytes, integrate with flt-1-regulated angiogenic molecular and cellular mechanisms to yield optimal vessel patterning for a given tissue.

The pericyte microenvironment during vascular development.

Vascular pericytes provide critical contributions to the formation and integrity of the blood vessel wall within the microcirculation. Pericytes maintain vascular stability and homeostasis by promoting endothelial cell junctions and depositing extracellular matrix (ECM) components within the vascular basement membrane, among other vital functions. As their importance in sustaining microvessel health within various tissues and organs continues to emerge, so does their role in a number of pathological conditions including cancer, diabetic retinopathy, and neurological disorders. Here, we review vascular pericyte contributions to the development and remodeling of the microcirculation, with a focus on the local microenvironment during these processes. We discuss observations of their earliest involvement in vascular development and essential cues for their recruitment to the remodeling endothelium. Pericyte involvement in the angiogenic sprouting context is also considered with specific attention to crosstalk with endothelial cells such as through signaling regulation and ECM deposition. We also address specific aspects of the collective cell migration and dynamic interactions between pericytes and endothelial cells during angiogenic sprouting. Lastly, we discuss pericyte contributions to mechanisms underlying the transition from active vessel remodeling to the maturation and quiescence phase of vascular development.

Excess vascular endothelial growth factor-A disrupts pericyte recruitment during blood vessel formation.

Pericyte investment into new blood vessels is essential for vascular development such that mis-regulation within this phase of vessel formation can contribute to numerous pathologies including arteriovenous and cerebrovascular malformations. It is critical therefore to illuminate how angiogenic signaling pathways intersect to regulate pericyte migration and investment. Here, we disrupted vascular endothelial growth factor-A (VEGF-A) signaling in ex vivo and in vitro models of sprouting angiogenesis, and found pericyte coverage to be compromised during VEGF-A perturbations. Pericytes had little to no expression of VEGF receptors, suggesting VEGF-A signaling defects affect endothelial cells directly but pericytes indirectly. Live imaging of ex vivo angiogenesis in mouse embryonic skin revealed limited pericyte migration during exposure to exogenous VEGF-A. During VEGF-A gain-of-function conditions, pericytes and endothelial cells displayed abnormal transcriptional changes within the platelet-derived growth factor-B (PDGF-B) and Notch pathways. To further test potential crosstalk between these pathways in pericytes, we stimulated embryonic pericytes with Notch ligands Delta-like 4 (Dll4) and Jagged-1 (Jag1) and found induction of Notch pathway activity but no changes in PDGF Receptor-ß (Pdgfrß) expression. In contrast, PDGFRß protein levels decreased with mis-regulated VEGF-A activity, observed in the effects on full-length PDGFRß and a truncated PDGFRß isoform generated by proteolytic cleavage or potentially by mRNA splicing. Overall, these observations support a model in which, during the initial stages of vascular development, pericyte distribution and coverage are indirectly affected by endothelial cell VEGF-A signaling and the downstream regulation of PDGF-B-PDGFRß dynamics, without substantial involvement of pericyte Notch signaling during these early stages.

Establishment and characterization of an embryonic pericyte cell line.

OBJECTIVE: Pericytes are specialized perivascular cells embedded within the basement membrane. These cells envelope the abluminal surface of endothelial cells and promote microvessel homeostasis. Recent discoveries of unique pericyte functions, particularly in neural tissues, underscore the need for overcoming existing challenges in establishing a functionally validated pericyte cell line. Here, we present methodologies for addressing these challenges as well as an embryonic pericyte cell line for use with in vitro and ex vivo experimental models. METHODS: We isolated an enriched population of NG2:DsRed+ pericytes from E12.5 mice. This pericyte cell line was compared to MEFs with respect to gene expression, cell morphology and migration, and engagement with endothelial cells during junction stabilization and angiogenesis. RESULTS: NG2+ pericytes displayed gene expression patterns, cell morphology, and 2D migration behaviors distinct from MEFs. In three different vessel formation models, pericytes from this line migrated to and incorporated into developing vessels. When co-cultured with HUVECs, these pericytes stimulated more robust VE-Cadherin junctions between HUVECs as compared to MEFs, as well as contributed to HUVEC organization into primitive vascular structures. CONCLUSIONS: Our data support use of this pericyte cell line in a broad range of models to further understand pericyte functionality during normal and pathological conditions.

Von Hippel-Lindau mutations disrupt vascular patterning and maturation via Notch.

Von Hippel-Lindau (VHL) gene mutations induce neural tissue hemangioblastomas, as well as highly vascularized clear cell renal cell carcinomas (ccRCCs). Pathological vessel remodeling arises from misregulation of HIFs and VEGF, among other genes. Variation in disease penetrance has long been recognized in relation to genotype. We show Vhl mutations also disrupt Notch signaling, causing mutation-specific vascular abnormalities, e.g., type 1 (null) vs. type 2B (murine G518A representing human R167Q). In conditional mutation retina vasculature, Vhl-null mutation (i.e., UBCCreER/+Vhlfl/fl) had little effect on initial vessel branching, but it severely reduced arterial and venous branching at later stages. Interestingly, this mutation accelerated arterial maturation, as observed in retina vessel morphology and aberrant α-smooth muscle actin localization, particularly in vascular pericytes. RNA sequencing analysis identified gene expression changes within several key pathways, including Notch and smooth muscle cell contractility. Notch inhibition failed to reverse later-stage branching defects but rescued the accelerated arterialization. Retinal vessels harboring the type 2B Vhl mutation (i.e., UBCCreER/+Vhlfl/2B) displayed stage-specific changes in vessel branching and an advanced progression toward an arterial phenotype. Disrupting Notch signaling in type 2B mutants increased both artery and vein branching and restored arterial maturation toward nonmutant levels. By revealing differential effects of the null and type 2B Vhl mutations on vessel branching and maturation, these data may provide insight into the variability of VHL-associated vascular changes - particularly the heterogeneity and aggressiveness in ccRCC vessel growth - and also suggest Notch pathway targets for treating VHL syndrome.