Modeling of Blood-Brain Barrier (BBB) Dysfunction and Immune Cell Migration Using Human BBB-on-a-Chip for Drug Discovery Research.

Blood-brain barrier (BBB) dysfunction is a key feature in neuroimmunological and neurodegenerative diseases. In this study, we developed a microfluidic human BBB-on-a-chip to model barrier dysfunction and immune cell migration using immortalized TY10 brain endothelial cells, pericytes, and astrocytes. It was found that immortalized TY10 brain endothelial cells developed a microvascular structure under flow. Pericytes were localized on the basal side surrounding the TY10 microvascular structure, showing an in vivo-like structure. Barrier integrity increased under co-culture with pericytes. In addition, both ethylenediaminetetraacetic acid (EDTA) and anti-Claudin-5 (CLDN5) neutralizing antibody caused a decrease in the transendothelial electrical resistance (TEER). EDTA caused the leakage of 20 kDa dextran, suggesting different effects on the BBB based on the mechanism of action, whereas anti-CLDN5 antibody did not cause leakage. In the tri-culture model, human T cells migrated through endothelial vessels towards basal C-X-C motif chemokine ligand 12 (CXCL12). The live-imaging analysis confirmed the extravasation of fluorescence-labelled T cells in a CXCL12-concentration- and time-dependent manner. Our BBB model had an in vivo-like structure and successfully represented barrier dysfunction and transendothelial T cell migration. In addition, our study suggests that the inhibition of CLDN5 attenuates the BBB in humans. This platform has various potential uses in relation to the BBB in both drug discovery research and in elucidating the mechanisms of central nervous system diseases.

Precise Modulation of Pericyte Dysfunction by a Multifunctional Nanoprodrug to Ameliorate Alzheimer's Disease.

Pericyte dysfunction severely undermines cerebrovascular integrity and exacerbates neurodegeneration in Alzheimer's disease (AD). However, pericyte-targeted therapy is a yet-untapped frontier for AD. Inspired by the elevation of vascular cell adhesion molecule-1 (VCAM-1) and reactive oxygen species (ROS) levels in pericyte lesions, we fabricated a multifunctional nanoprodrug by conjugating the hybrid peptide VLC, a fusion of the VCAM-1 high-affinity peptide VHS and the neuroprotective apolipoprotein mimetic peptide COG1410, to curcumin (Cur) through phenylboronic ester bond (VLC@Cur-NPs) to alleviate complex pericyte-related pathological changes. Importantly, VLC@Cur-NPs effectively homed to pericyte lesions via VLC and released their contents upon ROS stimulation to maximize their regulatory effects. Consequently, VLC@Cur-NPs markedly increased pericyte regeneration to form a positive feedback loop and thus improved neurovascular function and ultimately alleviated memory defects in APP/PS1 transgenic mice. We present a promising therapeutic strategy for AD that can precisely modulate pericytes and has the potential to treat other cerebrovascular diseases.

Growth hormone promotes myelin repair after chronic hypoxia via triggering pericyte-dependent angiogenesis.

White matter injury (WMI) causes oligodendrocyte precursor cell (OPC) differentiation arrest and functional deficits, with no effective therapies to date. Here, we report increased expression of growth hormone (GH) in the hypoxic neonatal mouse brain, a model of WMI. GH treatment during or post hypoxic exposure rescues hypoxia-induced hypomyelination and promotes functional recovery in adolescent mice. Single-cell sequencing reveals that Ghr mRNA expression is highly enriched in vascular cells. Cell-lineage labeling and tracing identify the GHR-expressing vascular cells as a subpopulation of pericytes. These cells display tip-cell-like morphology with kinetic polarized filopodia revealed by two-photon live imaging and seemingly direct blood vessel branching and bridging. Gain-of-function and loss-of-function experiments indicate that GHR signaling in pericytes is sufficient to modulate angiogenesis in neonatal brains, which enhances OPC differentiation and myelination indirectly. These findings demonstrate that targeting GHR and/or downstream effectors may represent a promising therapeutic strategy for WMI.

Polyethylene terephthalate (PET) micro- and nanoplastic particles affect the mitochondrial efficiency of human brain vascular pericytes without inducing oxidative stress.

The objective of this investigation was to evaluate the influence of micro- and nanoplastic particles composed of polyethylene terephthalate (PET), a significant contributor to plastic pollution, on human brain vascular pericytes. Specifically, we delved into their impact on mitochondrial functionality, oxidative stress, and the expression of genes associated with oxidative stress, ferroptosis and mitochondrial functions. Our findings demonstrate that the exposure of a monoculture of human brain vascular pericytes to PET particles in vitro at a concentration of 50 µg/ml for a duration of 3, 6 and 10 days did not elicit oxidative stress. Notably, we observed a reduction in various aspects of mitochondrial respiration, including maximal respiration, spare respiratory capacity, and ATP production in pericytes subjected to PET particles for 3 days, with a mitochondrial function recovery at 6 and 10 days. Furthermore, there were no statistically significant alterations in mitochondrial DNA copy number, or in the expression of genes linked to oxidative stress and ferroptosis, but an increase of the expression of the gene mitochondrial transcription factor A (TFAM) was noted at 3 days exposure. These outcomes suggest that, at a concentration of 50 µg/ml, PET particles do not induce oxidative stress in human brain vascular pericytes. Instead, at 3 days exposure, PET exposure impairs mitochondrial functions, but this is recovered at 6-day exposure. This seems to indicate a potential mitochondrial hormesis response (mitohormesis) is incited, involving the gene TFAM. Further investigations are warranted to explore the stages of mitohormesis and the potential consequences of plastics on the integrity of the blood-brain barrier and intercellular interactions. This research contributes to our comprehension of the potential repercussions of nanoplastic pollution on human health and underscores the imperative need for ongoing examinations into the exposure to plastic particles.

Shenqi Yizhi prescription prevents AßO-induced memory impairment in mice by regulating the contractility of brain pericytes.

BACKGROUND: Cerebral microcirculation disturbance manifested by decrease of cerebral blood flow (CBF) is one of early features of Alzheimer's disease (AD). Shenqi Yizhi prescription (SQYZ) is widely used in the treatment of AD. However, the effect of SQYZ on the early feature of AD is not clarified. PURPOSE: To explore the effect and mechanism of SQYZ on AD-like behavior from the perspective of early pathological features of AD. METHODS: The fingerprint of SQYZ was established by ultra-high-performance liquid chromatograph. The improvement effect of SQYZ on Aß1-42 Oligomer (AßO)-induced AD-like behavior of mice was evaluated by behavioral test. The changes of CBF were detected by laser doppler meter and laser speckle imaging. The pathological changes of the hippocampus were observed by HE staining and transmission electron microscope. The expressions of intercellular communication molecules were detected by western blotting or immunofluorescence staining. The content of platelet-derived growth factor-BB (PDGF-BB) was detected by ELISA. Finally, the core components of SQYZ were docked with platelet-derived growth factor receptor beta (PDGFRß) using AutoDock Vina software. RESULTS: The similarity of the components in SQYZ extracted from different batches of medicinal materials was higher than 0.9. SQYZ administration could improve AßO-induced memory impairment and CBF reduction. Compared with the sham group, the number of neurons in the hippocampi of AßO group was significantly reduced, and the microvessels were shrunken and deformed. By contrary, SQYZ administration mitigated those pathological changes. Compared with the sham mice, the expressions of CD31, N-cadherin, PDGFRß, glial fibrillary acidic protein, phosphorylation of focal adhesion kinase, integrin ß1, and integrin α5 in the hippocampi of AßO mice were significantly increased. However, SQYZ administration significantly reduced AßO-induced expression of those proteins. Interestingly, the effect of PDGFRß inhibitor, sunitinib demonstrated a consistent modulating effect as SQYZ. Finally, the brain-entering components of SQYZ, including ginsenoside Rg5, coptisine, cryptotanshinone, dihydrotanshinone IIA, stigmasterol, and tanshinone IIA had high binding force with PDGFRß, implicating PDGFRß as a potential target for SQYZ. CONCLUSIONS: Our data indicate that SQYZ improves CBF in AßO-triggered AD-like mice through inhibiting brain pericyte contractility, indicating the treatment potential of SQYZ for AD at the early stage.

Nanomodulators targeting endothelial WNT and pericytes to reversibly open the blood-tumor barrier for boosted brain tumor therapy.

The blood-brain barrier (BBB)/blood-tumor barrier (BTB) impedes brain entry of most brain-targeted drugs, whether they are water-soluble or hydrophobic. Endothelial WNT signaling and neoplastic pericytes maintain BTB low permeability by regulating tight junctions. Here, we proposed nitazoxanide (NTZ) and ibrutinib (IBR) co-loaded ICAM-1-targeting nanoparticles (NI@I-NPs) to disrupt the BTB in a time-dependent, reversible, and size-selective manner by targeting specific ICAM-1, inactivating WNT signaling and depleting pericytes in tumor-associated blood vessels in breast cancer brain metastases. At the optimal NTZ/IBR mass ratio (1:2), BTB opening reached the optimum effect at 48-72 h without any sign of intracranial edema and cognitive impairment. The combination of NI@I-NPs and chemotherapeutic drugs (doxorubicin and etoposide) extended the median survival of mice with breast cancer brain metastases. Targeting BTB endothelial WNT signaling and tumor pericytes via NI@I-NPs could open the BTB to improve chemotherapeutic efficiency against brain metastases.

C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Curcumin reduces blood-nerve barrier abnormalities and cytotoxicity to endothelial cells and pericytes induced by cisplatin.

BACKGROUND: Cisplatin is a platinum-based antineoplastic agent used to treat cancers of solid organs. Neuropathy is one of its major side effects, necessitating dose reduction or cessation. Previous studies suggested that cisplatin causes microvascular toxicity, including pericyte detachment. This study aimed to clarify whether these alterations occurred in the blood-nerve barrier (BNB) of capillaries after cisplatin treatment. MATERIALS AND METHODS AND RESULTS: Electron microscopic analysis of rat sciatic nerves with cisplatin neuropathy showed increased frequency and severity of pericyte detachment. Moreover, the vascular basement membrane did not tightly encircle around the endothelial cells and pericytes. Cultured human umbilical vein endothelial cells and human brain vascular pericytes showed reduced viability, increased caspase-3 activity and enhanced oxidative stress following cisplatin treatment. In addition, cisplatin decreased transendothelial electrical resistance (TEER) and the expression of the tight junction proteins occludin and zonula occludens-1. Curcumin, a polyphenol found in the root of Curcuma longa, had favourable effects on cisplatin neuropathy in previous work. Therefore, curcumin was tested to determine whether it had any effect on these abnormalities. Curcumin alleviated pericyte detachment, cytotoxicity, oxidative stress, TEER reduction and tight junction protein expression. CONCLUSIONS: These data indicate that cisplatin causes BNB disruption in the nerves and might result in neuropathy. Curcumin might improve neuropathy via the restoration of BNB. Whether alterations in the BNB occur and curcumin is effective in patients with cisplatin neuropathy remain to be investigated.

Lysophosphatidic acid stimulates pericyte migration via LPA receptor 1.

Lysophosphatidic acid (LPA) is a bioactive compound known to regulate various vascular functions. However, despite the fact that many vascular functions are regulated by peri-vascular cells such as pericytes, the effect of LPA on brain pericytes has not been fully evaluated. Thus, we designed this study to evaluate the effects of LPA on brain pericytes. These experiments revealed that while LPA receptors (LPARs) are expressed in cultured pericytes from mouse brains, LPA treatment does not influence the proliferation of these cells but does have a profound impact on their migration, which is regulated via the expression of LPAR1. LPAR1 expression was also detected in human pericyte culture and LPA treatment of these cells also induced migration. Taken together these findings imply that LPA-LPAR1 signaling is one of the key mechanisms modulating pericyte migration, which may help to control vascular function during development and repair processes.

Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex.

Multifaceted microglial functions in the developing brain, such as promoting the differentiation of neural progenitors and contributing to the positioning and survival of neurons, have been progressively revealed. Although previous studies have noted the relationship between vascular endothelial cells and microglia in the developing brain, little attention has been given to the importance of pericytes, the mural cells surrounding endothelial cells. In this study, we attempted to dissect the role of pericytes in microglial distribution and function in developing mouse brains. Our immunohistochemical analysis showed that approximately half of the microglia attached to capillaries in the cerebral walls. Notably, a magnified observation of the position of microglia, vascular endothelial cells and pericytes demonstrated that microglia were preferentially associated with pericytes that covered 79.8% of the total capillary surface area. Through in vivo pericyte depletion induced by the intraventricular administration of a neutralizing antibody against platelet-derived growth factor receptor (PDGFR)ß (clone APB5), we found that microglial density was markedly decreased compared with that in control antibody-treated brains because of their low proliferative capacity. Moreover, in vitro coculture of isolated CD11b+ microglia and NG2+PDGFRα- cells, which are mostly composed of pericytes, from parenchymal cells indicated that pericytes promote microglial proliferation via the production of soluble factors. Furthermore, pericyte depletion by APB5 treatment resulted in a failure of microglia to promote the differentiation of neural stem cells into intermediate progenitors. Taken together, our findings suggest that pericytes facilitate microglial homeostasis in the developing brains, thereby indirectly supporting microglial effects on neural progenitors.SIGNIFICANCE STATEMENT This study highlights the novel effect of pericytes on microglia in the developing mouse brain. Through multiple analyses using an in vivo pericyte depletion mouse model and an in vitro coculture study of isolated pericytes and microglia from parenchymal cells, we demonstrated that pericytes contribute to microglial proliferation and support microglia in efficiently promoting the differentiation of neural stem cells into intermediate progenitors. Our present data provide evidence that pericytes function not only in the maintenance of cerebral microcirculation and blood brain barrier (BBB) integrity but also in microglial homeostasis in the developing cerebral walls. These findings will expand our knowledge and help elucidate the mechanism of brain development both in healthy and disease conditions.

Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires the cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for the stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases the accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

Alteration of Extracellular Matrix Components in the Anterior Pituitary Gland of Neonatal Rats Induced by a Maternal Bisphenol A Diet during Pregnancy.

The extracellular matrix (ECM) plays crucial roles in the anterior pituitary gland via the mechanism of cell-ECM interaction. Since bisphenol A (BPA), a well-known endocrine disruptor, can cross through the placenta from mother to fetus and bind with estrogen receptors, cell populations in the neonatal anterior pituitary gland could be the target cells affected by this chemical. The present study treated maternal rats with 5000 µg/kg body weight of BPA daily throughout the pregnancy period and then investigated the changes in ECM-producing cells, i.e., pericytes and folliculostellate (FS) cells, including their ECM production in the neonatal anterior pituitary at Day 1. We found that pericytes and their collagen synthesis reduced, consistent with the increase in the number of FS cells that expressed several ECM regulators-matrix metalloproteinase (MMP) 9 and the tissue inhibitors of metalloproteinase (TIMP) family. The relative MMP9/TIMP1 ratio was extremely high, indicating that the control of ECM homeostasis was unbalanced. Moreover, transmission electron microscopy showed the unorganized cell cluster in the BPA-treated group. This study revealed that although the mother received BPA at the "no observed adverse effect" level, alterations in ECM-producing cells as well as collagen and the related ECM balancing genes occurred in the neonatal anterior pituitary gland.

SARS-CoV-2 deregulates the vascular and immune functions of brain pericytes via Spike protein.

Coronavirus disease 19 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via elusive mechanisms. SARS-CoV-2 infects host cells via the binding of viral Spike (S) protein to transmembrane receptor, angiotensin-converting enzyme 2 (ACE2). Although brain pericytes were recently shown to abundantly express ACE2 at the neurovascular interface, their response to SARS-CoV-2 S protein is still to be elucidated. Using cell-based assays, we found that ACE2 expression in human brain vascular pericytes was increased upon S protein exposure. Pericytes exposed to S protein underwent profound phenotypic changes associated with an elongated and contracted morphology accompanied with an enhanced expression of contractile and myofibrogenic proteins, such as α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). On the functional level, S protein exposure promoted the acquisition of calcium (Ca2+) signature of contractile ensheathing pericytes characterized by highly regular oscillatory Ca2+ fluctuations. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signaling pathway, which was potentiated by hypoxia, a condition associated with vascular comorbidities that exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely macrophage migration inhibitory factor (MIF). Using transgenic mice expressing the human ACE2 that recognizes S protein, we observed that the intranasal infection with SARS-CoV-2 rapidly induced hypoxic/ischemic-like pericyte reactivity in the brain of transgenic mice, accompanied with an increased vascular expression of ACE2. Moreover, we found that SARS-CoV-2 S protein accumulated in the intranasal cavity reached the brain of mice in which the nasal mucosa is deregulated. Collectively, these findings suggest that SARS-CoV-2 S protein impairs the vascular and immune regulatory functions of brain pericytes, which may account for vascular-mediated brain damage. Our study provides a better understanding for the mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.

Mechanism and potential treatment of the "no reflow" phenomenon after acute myocardial infarction: role of pericytes and GPR39.

The "no reflow" phenomenon, where the coronary artery is patent after treatment of acute myocardial infarction (AMI) but tissue perfusion is not restored, is associated with worse outcome. The mechanism of no reflow is unknown. We hypothesized that pericytes contraction, in an attempt to maintain a constant capillary hydrostatic pressure during reduced coronary perfusion pressure, causes capillary constriction leading to no reflow and that this effect is mediated through the orphan receptor, GPR39, present in pericytes. We created AMI (coronary occlusion followed by reperfusion) in GPR39 knock out mice and littermate controls. In a separate set of experiments, we treated wild-type mice undergoing coronary occlusion with vehicle or VC43, a specific inhibitor of GPR39, before reperfusion. We found that no reflow zones were significantly smaller in the GPR39 knockouts compared with controls. Both no reflow and infarct size were also markedly smaller in animals treated with VC43 compared with vehicle. Immunohistochemistry revealed greater capillary density and larger capillary diameter at pericyte locations in the GPR39-knockout and VC43-treated mice compared with controls. We conclude that GPR39-mediated pericyte contraction during reduced coronary perfusion pressure causes capillary constriction resulting in no reflow during AMI and that smaller no reflow zones in GPR39-knockout and VC43-treated animals are associated with smaller infarct sizes. These results elucidate the mechanism of no reflow in AMI, as well as providing a therapeutic pathway for the condition.NEW & NOTEWORTHY The mechanism of "no reflow" phenomenon, where the coronary artery is patent after treatment of acute myocardial infarction but tissue perfusion is not restored, is unknown. This condition is associated with worse outcome. Here, we show that GPR39-mediated pericyte contraction during reduced coronary perfusion pressure causes capillary constriction resulting in no reflow. Smaller no-reflow zones in GPR39-knockout animals and those treated with a GPR39 inhibitor are associated with smaller infarct size. These results could have important therapeutic implications.

Paricalcitol Improves Hypoxia-Induced and TGF-ß1-Induced Injury in Kidney Pericytes.

Recently, the role of kidney pericytes in kidney fibrosis has been investigated. This study aims to evaluate the effect of paricalcitol on hypoxia-induced and TGF-ß1-induced injury in kidney pericytes. The primary cultured pericytes were pretreated with paricalcitol (20 ng/mL) for 90 min before inducing injury, and then they were exposed to TGF-ß1 (5 ng/mL) or hypoxia (1% O2 and 5% CO2). TGF-ß1 increased α-SMA and other fibrosis markers but reduced PDGFRß expression in pericytes, whereas paricalcitol reversed the changes. Paricalcitol inhibited the TGF-ß1-induced cell migration of pericytes. Hypoxia increased TGF-ß1, α-SMA and other fibrosis markers but reduced PDGFRß expression in pericyte, whereas paricalcitol reversed them. Hypoxia activated the HIF-1α and downstream molecules including prolyl hydroxylase 3 and glucose transporter-1, whereas paricalcitol attenuated the activation of the HIF-1α-dependent molecules and TGF-ß1/Smad signaling pathways in hypoxic pericytes. The gene silencing of HIF-1α vanished the hypoxia-induced TGF-ß1, α-SMA upregulation, and PDGFRß downregulation. The effect of paricalcitol on the HIF-1α-dependent changes of fibrosis markers was not significant after the gene silencing of HIF-1α. In addition, hypoxia aggravated the oxidative stress in pericytes, whereas paricalcitol reversed the oxidative stress by increasing the antioxidant enzymes in an HIF-1α-independent manner. In conclusion, paricalcitol improved the phenotype changes of pericyte to myofibroblast in TGF-ß1-stimulated pericytes. In addition, paricalcitol improved the expression of fibrosis markers in hypoxia-exposed pericytes both in an HIF-1α-dependent and independent manner.

Estradiol Inhibits Human Brain Vascular Pericyte Migration Activity: A Functional and Transcriptomic Analysis.

Stroke is the third leading cause of mortality in women and it kills twice as many women as breast cancer. A key role in the pathophysiology of stroke plays the disruption of the blood-brain barrier (BBB) within the neurovascular unit. While estrogen induces vascular protective actions, its influence on stroke remains unclear. Moreover, experiments assessing its impact on endothelial cells to induce barrier integrity are non-conclusive. Since pericytes play an active role in regulating BBB integrity and function, we hypothesize that estradiol may influence BBB by regulating their activity. In this study using human brain vascular pericytes (HBVPs) we investigated the impact of estradiol on key pericyte functions known to influence BBB integrity. HBVPs expressed estrogen receptors (ER-α, ER-ß and GPER) and treatment with estradiol (10 nM) inhibited basal cell migration but not proliferation. Since pericyte migration is a hallmark for BBB disruption following injury, infection and inflammation, we investigated the effects of estradiol on TNFα-induced PC migration. Importantly, estradiol prevented TNFα-induced pericyte migration and this effect was mimicked by PPT (ER-α agonist) and DPN (ER-ß agonist), but not by G1 (GPR30 agonist). The modulatory effects of estradiol were abrogated by MPP and PHTPP, selective ER-α and ER-ß antagonists, respectively, confirming the role of ER-α and ER-ß in mediating the anti-migratory actions of estrogen. To delineate the intracellular mechanisms mediating the inhibitory actions of estradiol on PC migration, we investigated the role of AKT and MAPK activation. While estradiol consistently reduced the TNFα-induced MAPK and Akt phosphorylation, only the inhibition of MAPK, but not Akt, significantly abrogated the migratory actions of TNFα. In transendothelial electrical resistance measurements, estradiol induced barrier function (TEER) in human brain microvascular endothelial cells co-cultured with pericytes, but not in HBMECs cultured alone. Importantly, transcriptomics analysis of genes modulated by estradiol in pericytes showed downregulation of genes known to increase cell migration and upregulation of genes known to inhibit cell migration. Taken together, our findings provide the first evidence that estradiol modulates pericyte activity and thereby improves endothelial integrity.

Pericyte Insulin Receptors Modulate Retinal Vascular Remodeling and Endothelial Angiopoietin Signaling.

Pericytes regulate vascular development, stability, and quiescence; their dysfunction contributes to diabetic retinopathy. To explore the role of insulin receptors in pericyte biology, we created pericyte insulin receptor knockout mice (PIRKO) by crossing PDGFRß-Cre mice with insulin receptor (Insr) floxed mice. Their neonatal retinal vasculature exhibited perivenous hypervascularity with venular dilatation, plus increased angiogenic sprouting in superficial and deep layers. Pericyte coverage of capillaries was unaltered in perivenous and periarterial plexi, and no differences in vascular regression or endothelial proliferation were apparent. Isolated brain pericytes from PIRKO had decreased angiopoietin-1 mRNA, whereas retinal and lung angiopoietin-2 mRNA was increased. Endothelial phospho-Tie2 staining was diminished and FoxO1 was more frequently nuclear localized in the perivenous plexus of PIRKO, in keeping with reduced angiopoietin-Tie2 signaling. Silencing of Insr in human brain pericytes led to reduced insulin-stimulated angiopoietin-1 secretion, and conditioned media from these cells was less able to induce Tie2 phosphorylation in human endothelial cells. Hence, insulin signaling in pericytes promotes angiopoietin-1 secretion and endothelial Tie2 signaling and perturbation of this leads to excessive vascular sprouting and venous plexus abnormalities. This phenotype mimics elements of diabetic retinopathy, and future work should evaluate pericyte insulin signaling in this disease.

High-fat diet-induced diabetes leads to vascular alterations, pericyte reduction, and perivascular depletion of microglia in a 6-OHDA toxin model of Parkinson disease.

BACKGROUND: Diabetes has been recognized as a risk factor contributing to the incidence and progression of Parkinson's disease (PD). Although several hypotheses suggest a number of different mechanisms underlying the aggravation of PD caused by diabetes, less attention has been paid to the fact that diabetes and PD share pathological microvascular alterations in the brain. The characteristics of the interaction of diabetes in combination with PD at the vascular interface are currently not known. METHODS: We combined a high-fat diet (HFD) model of diabetes mellitus type 2 (DMT2) with the 6-OHDA lesion model of PD in male mice. We analyzed the association between insulin resistance and the achieved degree of dopaminergic nigrostriatal pathology. We further assessed the impact of the interaction of the two pathologies on motor deficits using a battery of behavioral tests and on microglial activation using immunohistochemistry. Vascular pathology was investigated histologically by analyzing vessel density and branching points, pericyte density, blood-brain barrier leakage, and the interaction between microvessels and microglia in the striatum. RESULTS: Different degrees of PD lesion were obtained resulting in moderate and severe dopaminergic cell loss. Even though the HFD paradigm did not affect the degree of nigrostriatal lesion in the acute toxin-induced PD model used, we observed a partial aggravation of the motor performance of parkinsonian mice by the diet. Importantly, the combination of a moderate PD pathology and HFD resulted in a significant pericyte depletion, an absence of an angiogenic response, and a significant reduction in microglia/vascular interaction pointing to an aggravation of vascular pathology. CONCLUSION: This study provides the first evidence for an interaction of DMT2 and PD at the brain microvasculature involving changes in the interaction of microglia with microvessels. These pathological changes may contribute to the pathological mechanisms underlying the accelerated progression of PD when associated with diabetes.

Eribulin induces tumor vascular remodeling through intussusceptive angiogenesis in a sarcoma xenograft model.

Eribulin is a novel microtubule inhibitor that, similar to other types of microtubule inhibitors, induces apoptosis by inhibiting the mitotic division of cells. Besides this direct effect on tumor cells, previous studies have shown that eribulin has the potential to induce tumor vascular remodeling in several different cancers; however, the mechanisms underlying this phenomenon remain unclear. In the present study, we aimed to elucidate whether eribulin is effective against synovial sarcoma, a relatively rare sarcoma that often affects adolescents and young adults, and to histologically investigate the microstructure of tumor vessels after the administration of eribulin. We found that eribulin exhibits potent antitumor activity against synovial sarcoma in a tumor xenograft model and that tumor vessels frequently have intervascular pillars, a hallmark of intussusceptive angiogenesis (IA), after the administration of eribulin. IA is a distinct form of angiogenesis that is involved in normal developmental processes as well as pathological conditions. Our data indicate that IA is potentially involved in eribulin-induced vascular remodeling and thereby suggest previously unacknowledged role of IA in regulating the tumor vasculature after eribulin administration.

Lenvatinib Targets PDGFR-ß Pericytes and Inhibits Synergy With Thyroid Carcinoma Cells: Novel Translational Insights.

CONTEXT: Pericyte populations abundantly express tyrosine kinases (eg, platelet-derived growth factor receptor-ß [PDGFR-ß]) and impact therapeutic response. Lenvatinib is a clinically available tyrosine kinase inhibitor that also targets PDGFR-ß. Duration of therapeutic response was shorter in patients with greater disease burden and metastasis. Patients may develop drug resistance and tumor progression. OBJECTIVES: Develop a gene signature of pericyte abundance to assess with tumor aggressiveness and determine both the response of thyroid-derived pericytes to lenvatinib and their synergies with thyroid carcinoma-derived cells. DESIGN: Using a new gene signature, we estimated the relative abundance of pericytes in papillary thyroid carcinoma (PTC) and normal thyroid (NT) TCGA samples. We also cocultured CD90+;PAX8- thyroid-derived pericytes and BRAFWT/V600E-PTC-derived cells to determine effects of coculture on paracrine communications and lenvatinib response. RESULTS: Pericyte abundance is significantly higher in BRAFV600E-PTC with hTERT mutations and copy number alterations compared with NT or BRAFWT-PTC samples, even when data are corrected for clinical-pathologic confounders. We have identified upregulated pathways important for tumor survival, immunomodulation, RNA transcription, cell-cycle regulation, and cholesterol metabolism. Pericyte growth is significantly increased by platelet-derived growth factor-BB, which activates phospho(p)-PDGFR-ß, pERK1/2, and pAKT. Lenvatinib strongly inhibits pericyte viability by down-regulating MAPK, pAKT, and p-p70S6-kinase downstream PDGFR-ß. Critically, lenvatinib significantly induces higher BRAFWT/V600E-PTC cell death when cocultured with pericytes, as a result of pericyte targeting via PDGFR-ß. CONCLUSIONS: This is the first thyroid-specific model of lenvatinib therapeutic efficacy against pericyte viability, which disadvantages BRAFWT/V600E-PTC growth. Assessing pericyte abundance in patients with PTC could be essential to selection rationales for appropriate targeted therapy with lenvatinib.