Pericytes In Mice Research Articles

Atp13a5 Marker Reveals Pericyte Specification in the Mouse Central Nervous System.

Perivascular mural cells including vascular smooth cells (VSMCs) and pericytes are integral components of the vascular system. In the central nervous system (CNS), pericytes are also indispensable for the blood-brain barrier (BBB), blood-spinal cord barrier, and blood-retinal barrier and play key roles in maintaining cerebrovascular and neuronal functions. However, the functional specifications of pericytes between CNS and peripheral organs have not been resolved at the genetic and molecular levels. Hence, the generation of reliable CNS pericyte-specific models and genetic tools remains very challenging. Here, we report a new CNS pericyte marker in mice. This putative cation-transporting ATPase 13A5 (Atp13a5) marker was identified through single-cell transcriptomics, based on its specificity to brain pericytes. We further generated a knock-in model with both tdTomato reporter and Cre recombinase. Using this model to trace the distribution of Atp13a5-positive pericytes in mice, we found that the tdTomato reporter reliably labels the CNS pericytes, including the ones in spinal cord and retina but not peripheral organs. Interestingly, brain pericytes are likely shaped by the developing neural environment, as Atp13a5-positive pericytes start to appear around murine embryonic day 15 (E15) and expand along the cerebrovasculature. Thus, Atp13a5 is a specific marker of CNS pericyte lineage, and this Atp13a5-based model is a reliable tool to explore the heterogeneity of pericytes and BBB functions in health and diseases.

Risk factors for severe COVID-19 disease increase SARS-CoV-2 infectivity of endothelial cells and pericytes.

Coronavirus disease 2019 (COVID-19) was initially considered a primarily respiratory disease but is now known to affect other organs including the heart and brain. A major route by which COVID-19 impacts different organs is via the vascular system. We studied the impact of apolipoprotein E (APOE) genotype and inflammation on vascular infectivity by pseudo-typed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses in mouse and human cultured endothelial cells and pericytes. Possessing the APOE4 allele or having existing systemic inflammation is known to enhance the severity of COVID-19. Using targeted replacement human APOE3 and APOE4 mice and inflammation induced by bacterial lipopolysaccharide (LPS), we investigated infection by SARS-CoV-2. Here, we show that infectivity was higher in murine cerebrovascular pericytes compared to endothelial cells and higher in cultures expressing APOE4. Furthermore, increasing the inflammatory state of the cells by prior incubation with LPS increased infectivity into human and mouse pericytes and human endothelial cells. Our findings provide insights into the mechanisms underlying severe COVID-19 infection, highlighting how risk factors such as APOE4 genotype and prior inflammation may exacerbate disease severity by augmenting the virus's ability to infect vascular cells.

Effects of repeated sleep deprivation on brain pericytes in mice

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-CreERT2/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.

ODP305 Differentiation Potential of Marginal Layer-Derived Adult Pituitary Stem/Progenitor Cells into Vascular Endothelial Cells and Pericytes in Mice

Abstract The anterior lobe (AL) of the pituitary gland consists five types of hormone-producing cells and fenestrated sinusoids (i. e., vascular endothelial cells and pericytes) that cooperate to maintain the physiological function of the gland. In addition, the population of cells located in pituitary stem/ progenitor cell niche, the marginal layer, which faces the residual lumen of the Rathke's pouch, serve to renew pituitary cells throughout life. We recently established a protocol for isolating marginal layer-derived adult pituitary stem/progenitor cells (MPSCs) by using differences in cell adhesion (1), and then showed differentiation potential into hormone-producing cells by subjecting MPSCs to three-dimensional culture. However, it is unclear whether MPSCs are capable of differentiating into all types of cells that constitute the pituitary gland. In this study, we performed DNA microarray analysis in MPSCs, which were proliferated by two-dimensional (2D) culture, for studying comprehensively gene expression profiles. Next, we investigated what kind of cells MPSCs would differentiate into. Comparing gene expression profiles of the AL and MPSCs, in MPSCs, 1776 genes were increased more than 4-fold and 1715 genes were decreased 4-fold compared to the AL (p<0. 05, respectively). The upregulated genes included pituitary stem/progenitor cell markers (ex. Sox2, Sox9, Klf4, Prrx1), niche markers (ex. Cdh1, Krt8), and cell surface antigens (ex. Cd9, Cd34, Cd44). On the other hand, most of the downregulated genes were differentiation markers (ex. Pou1f1, Gata2, Tbx19, Kdr). Next, immunofluorescent analysis revealed that most of the MPSCs were positive for CD34, the hematopoietic stem cell marker. After further 2D culture of MPSCs for 5-7days, vascular endothelial marker PECAM1-positive cells were detected by immunofluorescent analysis, but pericyte marker CSPG4-positive cell not. When TGFβ was applied to 2D culture of MPSCs, CSPG4-positive cells were detected instead of PECAM1-positive cells. Expression levels of Pecam1and Cspg4 were also examined by qPCR analysis, which corresponded to the results of immunofluorescent analysis. On the other hand, expression levels of anterior hormone genes were almost unchanged. These results have demonstrated that MPSCs could differentiate into vascular endothelial cells and pericytes. The present study suggests that pituitary stem/progenitor cells, which derived from the marginal layer, are a source of not only hormone-producing cells but also cells that constitute blood vessels. Reference: (1) Shintani and Higuchi, Stem Cell Res. 2021 Feb 2;52: 102223. Presentation: No date and time listed

Single Cell Transcriptomic Analysis Reveals Organ Specific Pericyte Markers and Identities.

Pericytes are mesenchymal-derived mural cells that wrap around capillaries and directly contact endothelial cells. Present throughout the body, including the cardiovascular system, pericytes are proposed to have multipotent cell-like properties and are involved in numerous biological processes, including regulation of vascular development, maturation, permeability, and homeostasis. Despite their physiological importance, the functional heterogeneity, differentiation process, and pathological roles of pericytes are not yet clearly understood, in part due to the inability to reliably distinguish them from other mural cell populations. Our study focused on identifying pericyte-specific markers by analyzing single-cell RNA sequencing data from tissue-specific mouse pericyte populations generated by the Tabula Muris Senis. We identified the mural cell cluster in murine lung, heart, kidney, and bladder that expressed either of two known pericyte markers, Cspg4 or Pdgfrb. We further defined pericytes as those cells that co-expressed both markers within this cluster. Single-cell differential expression gene analysis compared this subset with other clusters that identified potential pericyte marker candidates, including Kcnk3 (in the lung); Rgs4 (in the heart); Myh11 and Kcna5 (in the kidney); Pcp4l1 (in the bladder); and Higd1b (in lung and heart). In addition, we identified novel markers of tissue-specific pericytes and signaling pathways that may be involved in maintaining their identity. Moreover, the identified markers were further validated in Human Lung Cell Atlas and human heart single-cell RNAseq databases. Intriguingly, we found that markers of heart and lung pericytes in mice were conserved in human heart and lung pericytes. In this study, we, for the first time, identified specific pericyte markers among lung, heart, kidney, and bladder and reveal differentially expressed genes and functional relationships between mural cells.

TGF-β promotes pericyte-myofibroblast transition in subretinal fibrosis through the Smad2/3 and Akt/mTOR pathways

Subretinal fibrosis remains a major obstacle to the management of neovascular age-related macular degeneration. Choroidal pericytes were found to be a significant source of subretinal fibrosis, but the underlying mechanisms of pericyte-myofibroblast transition (PMT) remain largely unknown. The goal of this study was to explore the role and potential mechanisms by which PMT contributes to subretinal fibrosis. Choroidal neovascularization (CNV) was induced by laser photocoagulation in transgenic mice with the collagen1α1-green fluorescent protein (Col1α1-GFP) reporter, and recombinant adeno-associated virus 2 (rAAV2)-mediated TGF-β2 (rAAV2-TGF-β2) was administered intravitreally to further induce PMT. Primary mouse choroidal GFP-positive pericytes were treated with TGF-β2 in combination with siRNAs targeting Smad2/3, the Akt inhibitor MK2206 or the mTOR inhibitor rapamycin to examine cell proliferation, migration, and differentiation into myofibroblasts. The involvement of the Akt/mTOR pathway in PMT in subretinal fibrosis was further investigated in vivo. Intraocular TGF-β2 overexpression induced GFP-positive pericyte infiltration and PMT in subretinal fibrosis, which was mimicked in vitro. Knockdown of Smad2/3 or inhibition of Akt/mTOR decreased cell proliferation, PMT and migration in primary mouse pericytes. Combined inhibition of Smad2/3 and mTOR showed synergistic effects on attenuating α-smooth muscle actin (α-SMA) expression and cell proliferation. In mice with laser-induced CNV, the administration of the Akt/mTOR inhibitors suppressed pericyte proliferation and alleviated the severity of subretinal fibrosis. Our results showed that PMT plays a pivotal role in subretinal fibrosis, which was induced by TGF-β2 through the Smad2/3 and Akt/mTOR pathways. Thus, inhibiting PMT may be a novel strategy for the treatment of subretinal fibrosis.

The SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells: Implications for COVID-19 vascular research.

SummaryHumanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, so it is important to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the CNS, heart, and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed. In a number of other organs, pericytes do not express ACE2, including in the lung where ACE2 instead is expressed in bronchial epithelium and alveolar type II cells. The onset of ACE2 expression is organ specific: in bronchial epithelium already at birth, in brain pericytes before, and in heart pericytes after postnatal day 10.5. Establishing the vascular localization of ACE2 expression is central to correctly interpret data from modeling COVID-19 in the mouse and may shed light on the cause of vascular COVID-19 complications.

Validation of Specific and Reliable Genetic Tools to Identify, Label, and Target Cardiac Pericytes in Mice.

BackgroundIn the myocardium, pericytes are often confused with other interstitial cell types, such as fibroblasts. The lack of well‐characterized and specific tools for identification, lineage tracing, and conditional targeting of myocardial pericytes has hampered studies on their role in heart disease. In the current study, we characterize and validate specific and reliable strategies for labeling and targeting of cardiac pericytes.Methods and ResultsUsing the neuron‐glial antigen 2 (NG2)DsRed reporter line, we identified a large population of NG2+ periendothelial cells in mouse atria, ventricles, and valves. To examine possible overlap of NG2+ mural cells with fibroblasts, we generated NG2DsRed; platelet‐derived growth factor receptor (PDGFR) αEGFP pericyte/fibroblast dual reporter mice. Myocardial NG2+ pericytes and PDGFRα+ fibroblasts were identified as nonoverlapping cellular populations with distinct transcriptional signatures. PDGFRα+ fibroblasts expressed high levels of fibrillar collagens, matrix metalloproteinases, tissue inhibitor of metalloproteinases, and genes encoding matricellular proteins, whereas NG2+ pericytes expressed high levels of Pdgfrb, Adamts1, and Vtn. To validate the specificity of pericyte Cre drivers, we crossed these lines with PDGFRαEGFP fibroblast reporter mice. The constitutive NG2Cre driver did not specifically track mural cells, labeling many cardiomyocytes. However, the inducible NG2CreER driver specifically traced vascular mural cells in the ventricle and in the aorta, without significant labeling of PDGFRα+ fibroblasts. In contrast, the inducible PDGFRβCreER line labeled not only mural cells but also the majority of cardiac and aortic fibroblasts.ConclusionsFibroblasts and pericytes are topographically and transcriptomically distinct populations of cardiac interstitial cells. The inducible NG2CreER driver optimally targets cardiac pericytes; in contrast, the inducible PDGFRβCreER line lacks specificity.

P0098CHANGES OF PERICYTE IN AGING KIDNEY

Abstract Background and Aims In aging kidney, oxidative stress and inflammation lead to a decrease in renal function. Recent studies related to the aging process of the kidneys have shown that pericyte is a progenitor cell of myofibroblast, and the relationship between renal fibrosis and pericyte has been reported. In this study, we investigated the pericytes changes in aging kidneys and mechanism of aging-related pericyte changes. Method In vivo study, 2, 24-months-old mice and 18-month-old mice treated with pentoxifylline for 6 months were analyzed with western blot. And renal biopsy from KT donors were analyzed. Donors under age 25 were assigned to young group and donors over age 60 to old group. Results Changes of pericytes in mice and human renal tissues were consistent with those in the aging model. In the old age group, pericytes were decreased and interstitial fibrosis was increased. Western blot analysis in mice kidney revealed that IL-1β, IL-6 and TNF-α were significantly increased in old age and significantly decreased in pentoxifylline group. Angiopoietin 2 was increased in old age group and decreased in pentoxifylline group. Conclusion In aging kidney, pericytes have been shown to exhibit numerical reduction and loss of perivascular location as in the aging mice model. These changes in pericytes are thought to be induced by angiopoietin 2 excretion.

Intrinsic Myogenic Potential of Skeletal Muscle-Derived Pericytes from Patients with Myotonic Dystrophy Type 1

Pericytes are multipotent, vessel-associated progenitors that exhibit high proliferative capacity, can cross the blood-muscle barrier, and have the ability to home to muscle tissue and contribute to myogenesis. Consequently, pericyte-based therapies hold great promise for muscular dystrophies. A complex multi-system disorder exhibiting muscular dystrophy for which pericytes might be a valuable cell source is myotonic dystrophy type 1 (DM1). DM1 is caused by an unstable (CTG)n repeat in the DMPK gene and characterized by skeletal muscle weakness, muscle wasting, and myotonia. We have successfully isolated alkaline phosphatase-positive pericytes from skeletal muscle of DM1 patients and a transgenic mouse model. Intranuclear (CUG)n RNA foci, a pathogenic DM1 hallmark, were identified in human and mouse pericytes. Notably, pericytes from DM1 patients maintained similar growth parameters and innate myogenic characteristics in vitro compared to cells from unaffected controls. Our in vitro results thus demonstrate the potential of pericytes to ameliorate muscle features in DM1 in a therapeutic setting.

Blood-brain barrier-associated pericytes internalize and clear aggregated amyloid-β42 by LRP1-dependent apolipoprotein E isoform-specific mechanism

BackgroundClearance at the blood-brain barrier (BBB) plays an important role in removal of Alzheimer’s amyloid-β (Aβ) toxin from brain both in humans and animal models. Apolipoprotein E (apoE), the major genetic risk factor for AD, disrupts Aβ clearance at the BBB. The cellular and molecular mechanisms, however, still remain unclear, particularly whether the BBB-associated brain capillary pericytes can contribute to removal of aggregated Aβ from brain capillaries, and whether removal of Aβ aggregates by pericytes requires apoE, and if so, is Aβ clearance on pericytes apoE isoform-specific.MethodsWe performed immunostaining for Aβ and pericyte biomarkers on brain capillaries (< 6 μm in diameter) on tissue sections derived from AD patients and age-matched controls, and APPSwe/0 mice and littermate controls. Human Cy3-Aβ42 uptake by pericytes was studied on freshly isolated brain slices from control mice, pericyte LRP1-deficient mice (Lrplox/lox;Cspg4-Cre) and littermate controls. Clearance of aggregated Aβ42 by mouse pericytes was studied on multi-spot glass slides under different experimental conditions including pharmacologic and/or genetic inhibition of the low density lipoprotein receptor related protein 1 (LRP1), an apoE receptor, and/or silencing mouse endogenous Apoe in the presence and absence of human astrocyte-derived lipidated apoE3 or apoE4. Student’s t-test and one-way ANOVA followed by Bonferroni's post-hoc test were used for statistical analysis.ResultsFirst, we found that 35% and 60% of brain capillary pericytes accumulate Aβ in AD patients and 8.5-month-old APPSw/0 mice, respectively, compared to negligible uptake in controls. Cy3-Aβ42 species were abundantly taken up by pericytes on cultured mouse brain slices via LRP1, as shown by both pharmacologic and genetic inhibition of LRP1 in pericytes. Mouse pericytes vigorously cleared aggregated Cy3-Aβ42 from multi-spot glass slides via LRP1, which was inhibited by pharmacologic and/or genetic knockdown of mouse endogenous apoE. Human astrocyte-derived lipidated apoE3, but not apoE4, normalized Aβ42 clearance by mouse pericytes with silenced mouse apoE.ConclusionsOur data suggest that BBB-associated pericytes clear Aβ aggregates via an LRP1/apoE isoform-specific mechanism. These data support the role of LRP1/apoE interactions on pericytes as a potential therapeutic target for controlling Aβ clearance in AD.

Human adipose tissue-derived stromal cells act as functional pericytes in mice and suppress high-glucose-induced proinflammatory activation of bovine retinal endothelial cells

Aims/hypothesisThe immunomodulatory capacity of adipose tissue-derived stromal cells (ASCs) is relevant for next-generation cell therapies that aim to reverse tissue dysfunction such as that caused by diabetes. Pericyte dropout from retinal capillaries underlies diabetic retinopathy and the subsequent aberrant angiogenesis.MethodsWe investigated the pericytic function of ASCs after intravitreal injection of ASCs in mice with retinopathy of prematurity as a model for clinical diabetic retinopathy. In addition, ASCs influence their environment by paracrine signalling. For this, we assessed the immunomodulatory capacity of conditioned medium from cultured ASCs (ASC-Cme) on high glucose (HG)-stimulated bovine retinal endothelial cells (BRECs).ResultsASCs augmented and stabilised retinal angiogenesis and co-localised with capillaries at a pericyte-specific position. This indicates that cultured ASCs exert juxtacrine signalling in retinal microvessels. ASC-Cme alleviated HG-induced oxidative stress and its subsequent upregulation of downstream targets in an NF-κB dependent fashion in cultured BRECs. Functionally, monocyte adhesion to the monolayers of activated BRECs was also decreased by treatment with ASC-Cme and correlated with a decline in expression of adhesion-related genes such as SELE, ICAM1 and VCAM1.Conclusions/interpretationThe ability of ASC-Cme to immunomodulate HG-challenged BRECs is related to the length of time for which ASCs were preconditioned in HG medium. Conditioned medium from ASCs that had been chronically exposed to HG medium was able to normalise the HG-challenged BRECs to normal glucose levels. In contrast, conditioned medium from ASCs that had been exposed to HG medium for a shorter time did not have this effect. Our results show that the manner of HG preconditioning of ASCs dictates their immunoregulatory properties and thus the potential outcome of treatment of diabetic retinopathy.

Primary mouse brain pericytes isolated from transgenic Alzheimer mice spontaneously differentiate into a CD11b+ microglial-like cell type in vitro.

Alzheimer's disease (AD) is characterized by amyloid-β plaques, tau pathology and vascular impairment including pericyte damage. Pericytes are perivascular cells of the blood-brain barrier and can differentiate into different cell types in vitro including microglia. The aim of the present study is to explore if primary mouse brain pericytes isolated and cultured from transgenic AD (APP_SweDI) mice can differentiate into CD11b+ (integrin alpha M) microglia in vitro. We show that primary pericytes (passage 5) isolated from wildtype C57BL6 mice differentiated into CD11b+ microglia (Type B, >90%), when exposed to a differentiation factor mix of FGF-2, cAMP and fibronectin. This differentiation was time-dependent and seen as a large 80 kDa CD11b fragment (days 1-8) and a smaller 50 kDA CD11b fragment (>4 days). These pericytes did not differentiate into neurons, astroglia or oligodendroglia. However, pericytes isolated from transgenic AD mice differentiated into CD11b+ microglia (Type A, <10%) without addition of exogenous differentiation factors, displayed moderate Iba1+ immunostaining and phagocytic activity, but were still positive for PDGFRβ. In conclusion, we show for the first time that primary mouse pericytes from AD mice have the potential to spontanously differentiate in vitro into a CD11b+ microglial-like (Type A) cell type, but we do not provide evidence that these pericytic microglia display a full active microglial cell.

Effects of basic fibroblast growth factor on pericytes in mice with traumatic brain injury

Objective To investigate the effects of basic fibroblast growth factor (bFGF) on pericytes in the blood brain barrier at acute stage after traumatic brain injury (TBI) in mice. Methods A total of 90 mice with a C57BL/6 background were randomly divided into sham group, TBI group, and TBI+ bFGF group, with 30 rats per group. The models of moderate TBl were established using the controlled cortical impactor. After 24 hours, the changes of nerve function were evaluated by Garcia neurological score. Each mouse received an intraperitoneal injection of Evans blue dye for measuring the permeability of blood brain barrier. Western blot was used to test the related indices of pericytes after the cerebral cortex was quickly dissected: platelet-derived growth factor receptor beta (PDGFR-β), aminopeptidase N(CD13), desmin, neurogliocyte 2 (NG2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Paraffin sections were prepared for HE staining and morphological changes were observed. Immunofluorescence assay was used to test the related indices of pericytes: PDGFR-β, CD13, and cell surface glycoprotein MUC18 (CD146). Results Garcia neurological score revealed that the score in TBI group was significantly decreased compared with that in sham group (P<0.01), but the score of TBI+ bFGF group was significantly increased compared with that of TBI group (P<0.05). Permeability of blood brain barrier in TBI group was significantly increased compared with that in sham group (P<0.01), but in TBI+ bFGF group this parameter significantly reduced compared with that in TBI group (P<0.01). Western blot analysis revealed that the expressions of PDGFR-β, CD13, desmin, NG2 proteins in TBI group were significantly decreased compared with those in sham group (P<0.05), while the expressions of PDGFR-β, CD13, desmin, NG2 proteins in TBI+ bFGF group were significantly increased compared with those in TBI group (P<0.05). HE staining revealed injury of brain parenchyma in TBI group was the severest compared with both sham group and TBI+ bFGF group. Immunofluorescence staining results revealed that the proteins expressions of PDGFR-β, CD13, and CD146 in TBI group were significantly decreased compared with those in sham group (all P<0.01), and those in TBI+ bFGF group were significantly increased compared with those in TBI group (all P<0.05). Conclusions bFGF can prevent pericyte death via protecting its proteins to conserve blood-brain barrier. bFGF can also significantly ameliorate the injury of brain parenchyma. Key words: Brain injuries; Fibroblast growth factor 2; Pericyte

Abstract 4933: Desmin+pericyte subpopulations correlated with blood-tumor barrier permeability in brain metastases of breast cancer

Abstract Breast cancer brain metastases remain incurable. The blood-brain barrier (BBB) is a multicellular dynamic structure regulating exchanges between the blood and the central nervous system. As cancer cells colonize the brain, the BBB evolves into a blood-tumor barrier (BTB). The BTB limits compound penetration and therefore contributes to poor efficacy of chemotherapy. While the BBB has been well characterized in developmental and neurodegenerative disease studies, the BTB composition remains unknown. We have characterized the BTB in three model systems of brain metastasis of breast cancer developed in the laboratory: a triple negative (231-BR6), and two HER2 overexpressing (SUM190-BR3, JIMT-1-BR3) subtypes. Using Texas Red dextran (TRD) as a marker of permeability and quantitative immunofluorescence staining, we analyzed the cellular and molecular composition of: 1) unaltered BBB vs. BTB, and 2) BTB in highly permeable metastases vs. BTB in poorly permeable metastases. The BTB developed from the BBB in a series of alterations, including a neuroinflammatory reaction with astrogliosis, endothelial cell dilation, increased VEGF, reduced astrocyte endfoot polarity, and decrease in PDGFR+ pericytes. Only 10% of the metastatic lesions harbored a profound TRD exudation, which correlated with paclitaxel efficacy. We hypothesized that specific cellular and molecular changes account for the heterogeneity and increase in TRD diffusion. When metastases with relatively low- and high-TRD diffusion were compared, highly permeable metastases correlated with an increased expression of desmin+ pericytes in three models (231-BR6 p=0.0002; JIMT-1-BR3 p=0.004; SUM190-BR3 p=0.008) and a decrease in CD13+ pericytes in two model systems (231-BR6 p=0.014; JIMT-1-BR3 p=0.002). Decreased expression of laminin α2 in the parenchymal basement membrane (231-BR6 p=0.001; JIMT-1-BR3 p=0.049; SUM190-BR3 p=0.023) were associated with higher permeability. Desmin+ pericytes have been associated with pathological conditions such as fibrosis and spinal cord injury. Seven over nine human craniotomy specimens were positive for Desmin staining, validating clinically the relevance of our findings. We subsequently hypothesized that the desmin+ pericyte subpopulation functionally contributes to increased permeability. Desmin+ pericytes were produced in vitro by co-culturing primary mouse pericytes with astrocytes. When desmin+ or CD13+ pericytes were added to in vitro transendothelial electrical resistance (TEER) models of the BBB, the desmin+ pericytes exhibited less resistance, indicative of higher permeability. The data suggest that desmin+ pericytes may facilitate the permeability of the BTB. These studies show that the BTB in brain metastasis model systems involves consistent molecular changes. These data may identify new strategies to selectively permeabilize the BTB and enhance chemotherapeutic efficacy. Citation Format: Brunilde Gril, L. Tiffany Lyle, Paul R. Lockman, Chris E. Adkins, Afroz Shareef Mohammad, Emily Sechrest, Emily Hua, Diane Palmieri, David J. Liewehr, Seth M. Steinberg, Wojciech Kloc, Ewa Izycka-Swieszewska, Renata Duchnowska, Nayyar Naema, Priscilla K. Brastianos, Patricia S. Steeg. Desmin+pericyte subpopulations correlated with blood-tumor barrier permeability in brain metastases of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4933. doi:10.1158/1538-7445.AM2017-4933

Platelet-derived Growth Factor Receptor-beta is Differentially Regulated in Primary Mouse Pericytes and Brain Slices.

Pericytes are perivascular cells and have heterogenous roles in the brain, such as controlling blood flow and entry of immune cells or regulating the blood-brain barrier. Platelet-derived growth factor (PDGF) receptor-beta (PDGFRβ) is highly expressed in pericytes, representing the most selective biomarker. The aim of the present study was to culture primary mouse pericytes and to determine the expression pattern by Western Blot as well as immunostainings. We will study the effects of different exogenous stimuli (such as transforming growth factor-β (TGFβ1), PDGF-BB, oxygendeprivation, beta-amyloid or serumfree conditions) on the different pericyte markers. Using Western Blot analyses, we show that PDGFRβ is selectively expressed in pericytes as a 160 kDa protein. Nestin, although not exclusively specific, is also expressed by pericytes, but markedly downregulated under serum-free conditions. PDGF-BB and oxygen-deprivation dramatically reduced PDGFRβ expression, while TGFβ1 increased its expression. The expression of PDGFRβ was intracellular as shown by confocal microscopy. Using Western Blot analyses, we demonstrate that pericytes also contain a 100 kDa PDGFRβ protein. However, in contrast to cortex brain slices, pericytes do not express a phosphorylated (Y740) isoform. Interestingly, PDGF-BB markedly reduced the 160 kDa isoform of PDGFRβ. In conclusion, our data show a detailed expression of different forms of PDGFRβ in primary pericytes, which is different to brain slices. However, we suggest that PDGFRβ is a highly selective marker for pericytes.

Expression Patterns and Phenotypic Changes Regarding Stemness in Brain Pericytes in Health and Disease

Objective: Brain pericytes (PCs), which exist near endothelial cells, function in processes including maintenance of the blood–brain barrier and as stem cells. However, the relationship between expression patterns and phenotypic changes regarding stemness in brain PCs remains unclear. Thus, we investigated the properties of brain PCs in healthy and diseased mice. Methods: We examined the expression of representative pericytic markers, such as neuron-glial antigen 2 (NG2) and alpha smooth muscle actin (αSMA), and of the stem cell marker Nestin, in developing and pathologic mouse brains. Results: Brain PCs expressed NG2 and αSMA during the embryonic and postnatal stages of development, but rarely during adulthood. Although brain PCs exhibited Nestin expression during early development, it was scarce during adulthood, in parallel with a decrease in pericytic marker expression. However, upon brain injury following ischemic stroke, NG2 and αSMA were significantly induced in PCs in adult mice, concomitant with upregulated Nestin expression. Conclusion: The expression of markers in brain PCs differs during development and between normal and pathologic conditions in addition to traits such as stemness. An understanding of these expression profiles will be useful for PC-based stem cell therapies in the future.

Regional Blood Flow in the Normal and Ischemic Brain Is Controlled by Arteriolar Smooth Muscle Cell Contractility and Not by Capillary Pericytes

The precise regulation of cerebral blood flow is critical for normal brain function, and its disruption underlies many neuropathologies. The extent to which smooth muscle-covered arterioles or pericyte-covered capillaries control vasomotion during neurovascular coupling remains controversial. We found that capillary pericytes in mice and humans do not express smooth muscle actin and are morphologically and functionally distinct from adjacent precapillary smooth muscle cells (SMCs). Using optical imaging we investigated blood flow regulation at various sites on the vascular tree in living mice. Optogenetic, whisker stimulation, or cortical spreading depolarization caused microvascular diameter or flow changes in SMC but not pericyte-covered microvessels. During early stages of brain ischemia, transient SMC but not pericyte constrictions were amajor cause of hypoperfusion leading to thrombosis and distal microvascular occlusions. Thus, capillary pericytes are not contractile, and regulation of cerebral blood flow in physiological and pathological conditions is mediated by arteriolar SMCs.

Inhibition of APE1/Ref-1 redox activity with APX3330 blocks retinal angiogenesis in vitro and in vivo

This study examines the role of APE1/Ref-1 in the retina and its potential as a therapeutic target for inhibiting retinal angiogenesis. APE1/Ref-1 expression was quantified by Western blot. The role of APE1/Ref-1 redox function in endothelial cell in vitro angiogenesis was examined by treating retinal vascular endothelial cells (RVECs) with APX3330, a small molecule inhibitor of APE1/Ref-1 redox activity. In vitro methods included a proliferation assay, a transwell migration assay, a Matrigel tube formation assay, and a Real-Time Cell Analysis (RTCA) using the xCELLigence System. In vivo functional studies of APE1/Ref-1 were carried out by treating very low density lipoprotein (VLDL) receptor knockout mice (Vldlr−/−) with intravitreal injection of APX3330, and subsequent measurement of retinal angiomatous proliferation (RAP)-like neovascularization for one week. APE1/Ref-1 was highly expressed in the retina and in RVECs and pericytes in mice. APX3330 (1–10μM) inhibited proliferation, migration and tube formation of RVECs in vitro in a dose-dependent manner. Vldlr−/− RVECs were more sensitive to APX3330 than wild-type RVECs. In Vldlr−/− mice, a single intravitreal injection of APX3330 at the onset of RAP-like neovascularization significantly reduced RAP-like neovascularization development. APE1/Ref-1 is expressed in retinal vascular cells. APX3330 inhibits RVEC angiogenesis in vitro and significantly reduces RAP-like neovascularization in Vldlr−/− mice. These data support the conclusion that APE1/Ref-1 redox function is required for retinal angiogenesis. Thus, APE1/Ref-1 may have potential as a therapeutic target for treating neovascular age-related macular degeneration and other neovascular diseases.