Adipose stem cells are sexually dimorphic cells with dual roles as preadipocytes and resident fibroblasts.

Cell identities are defined by intrinsic transcriptional networks and spatio-temporal environmental factors. Here, we explored multiple factors that contribute to the identity of adipose stem cells, including anatomic location, microvascular neighborhood, and sex. Our data suggest that adipose stem cells serve a dual role as adipocyte precursors and fibroblast-like cells that shape the adipose tissue's extracellular matrix in an organotypic manner. We further find that adipose stem cells display sexual dimorphism regarding genes involved in estrogen signaling, homeobox transcription factor expression and the renin-angiotensin-aldosterone system. These differences could be attributed to sex hormone effects, developmental origin, or both. Finally, our data demonstrate that adipose stem cells are distinct from mural cells, and that the state of commitment to adipogenic differentiation is linked to their anatomic position in the microvascular niche. Our work supports the importance of sex and microvascular function in adipose tissue physiology.

A single-cell transcriptomic inventory of murine smooth muscle cells.

Smooth muscle cells (SMCs) execute important physiological functions in numerous vital organ systems, including the vascular, gastrointestinal, respiratory, and urogenital tracts. SMC differ morphologically and functionally at these different anatomical locations, but the molecular underpinnings of the differences remain poorly understood. Here, using deep single-cell RNA sequencing combined with in situ gene and protein expression analysis in four murine organs-heart, aorta, lung, and colon-we identify a molecular basis for high-level differences among vascular, visceral, and airway SMC, as well as more subtle differences between, for example, SMC in elastic and muscular arteries and zonation of elastic artery SMC along the direction of blood flow. Arterial SMC exhibit extensive organotypic heterogeneity, whereas venous SMC are similar across organs. We further identify a specific SMC subtype within the pulmonary vasculature. This comparative SMC cross-organ resource offers insight into SMC subtypes and their specific functions.

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

Humanized 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.

RGS5 Determines Neutrophil Migration in the Acute Inflammatory Phase of Bleomycin-Induced Lung Injury.

The regulator of G protein signaling (RGS) represents a widespread system of controllers of cellular responses. The activities of the R4 subfamily of RGSs have been elucidated in allergic pulmonary diseases. However, the R4 signaling in other inflammatory lung diseases, with a strong cellular immune response, remained unexplored. Thus, our study aimed to discern the functional relevance of the R4 family member, RGS5, as a potential modulating element in this context. Gene profiling of the R4 subfamily showed increased RGS5 expression in human fibrosing lung disease samples. In line with this, RGS5 was markedly increased in murine lungs following bleomycin injury. RGS knock-out mice (RGS-/-) had preserved lung function while control mice showed significant combined ventilatory disorders three days after bleomycin application as compared to untreated control mice. Loss of RGS5 was associated with a significantly reduced neutrophil influx and tissue myeloperoxidase expression. In the LPS lung injury model, RGS5-/- mice also failed to recruit neutrophils into the lung, which was accompanied by reduced tissue myeloperoxidase levels after 24 h. Our in-vitro assays showed impaired migration of RGS5-/- neutrophils towards chemokines despite preserved Ca2+ signaling. ERK dephosphorylation might play a role in reduced neutrophil migration in our model. As a conclusion, loss of RGS5 preserves lung function and attenuates hyperinflammation in the acute phase of bleomycin-induced pulmonary fibrosis and LPS-induced lung injury. Targeting RGS5 might alleviate the severity of exacerbations in interstitial lung diseases.

Retinoic acid receptor responder1 promotes development of glomerular diseases via the Nuclear Factor-κB signaling pathway.

Inflammatory pathways are activated in most glomerular diseases but molecular mechanisms driving them in kidney tissue are poorly known. We identified retinoic acid receptor responder 1 (Rarres1) as a highly podocyte-enriched protein in healthy kidneys. Studies in podocyte-specific knockout animals indicated that Rarres1 was not needed for the normal development or maintenance of the glomerulus filtration barrier and did not modulate the outcome of kidney disease in a model of glomerulonephritis. Interestingly, we detected an induction of Rarres1 expression in glomerular and peritubular capillary endothelial cells in IgA and diabetic kidney disease, as well as in ANCA-associated vasculitis. Analysis of publicly available RNA data sets showed that the induction of Rarres1 expression was a common molecular mechanism in chronic kidney diseases. A conditional knock-in mouse line, overexpressing Rarres1 specifically in endothelial cells, did not show any obvious kidney phenotype. However, the overexpression promoted the progression of kidney damage in a model of glomerulonephritis. In line with this, conditional knock-out mice, lacking Rarres1 in endothelial cells, were partially protected in the disease model. Mechanistically, Rarres1 promoted inflammation and fibrosis via transcription factor Nuclear Factor-κB signaling pathway by activating receptor tyrosine kinase Axl. Thus, induction of Rarres1 expression in endothelial cells is a prevalent molecular mechanism in human glomerulopathies and this seems to have a pathogenic role in driving inflammation and fibrosis via the Nuclear Factor-κB signaling pathway.

Publisher Correction: Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination.

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.

Parenchymal pericytes are not the major contributor of extracellular matrix in the fibrotic scar after stroke in male mice.

Scar formation after injury of the brain or spinal cord is a common event. While glial scar formation by astrocytes has been extensively studied, much less is known about the fibrotic scar, in particular after stroke. Platelet-derived growth factor receptor ß-expressing (PDGFRß+ ) pericytes have been suggested as a source of the fibrotic scar depositing fibrous extracellular matrix (ECM) proteins after detaching from the vessel wall. However, to what extent these parenchymal PDGFRß+ cells contribute to the fibrotic scar and whether targeting these cells affects fibrotic scar formation in stroke is still unclear. Here, we utilize male transgenic mice that after a permanent middle cerebral artery occlusion stroke model have a shift from a parenchymal to a perivascular location of PDGFRß+ cells due to the loss of regulator of G-protein signaling 5 in pericytes. We find that only a small fraction of parenchymal PDGFRß+ cells co-label with type I collagen and fibronectin. Consequently, a reduction in parenchymal PDGFRß+ cells by ca. 50% did not affect the overall type I collagen or fibronectin deposition after stroke. The redistribution of PDGFRß+ cells to a perivascular location, however, resulted in a reduced thickening of the vascular basement membrane and changed the temporal dynamics of glial scar maturation after stroke. We demonstrate that parenchymal PDGFRß+ cells are not the main contributor to the fibrotic ECM, and therefore targeting these cells might not impact on fibrotic scar formation after stroke.

Formation and metabolism of oxysterols and cholestenoic acids found in the mouse circulation: Lessons learnt from deuterium-enrichment experiments and the CYP46A1 transgenic mouse.

While the presence and abundance of the major oxysterols and cholestenoic acids in the circulation is well established, minor cholesterol metabolites may also have biological importance and be of value to investigate. In this study by observing the metabolism of deuterium-labelled cholesterol in the pdgfbret/ret mouse, a mouse model with increased vascular permeability in brain, and by studying the sterol content of plasma from the CYP46A1 transgenic mouse overexpressing the human cholesterol 24S-hydroxylase enzyme we have been able to identify a number of minor cholesterol metabolites found in the circulation, make approximate-quantitative measurements and postulate pathways for their formation. These "proof of principle" data may have relevance when using mouse models to mimic human disease and in respect of the increasing possibility of treating human neurodegenerative diseases with pharmaceuticals designed to enhance the activity of CYP46A1 or by adeno-associated virus delivery of CYP46A1.

Regulator of G-protein signaling 5 regulates the shift from perivascular to parenchymal pericytes in the chronic phase after stroke.

Poststroke recovery requires multiple repair mechanisms, including vascular remodeling and blood-brain barrier (BBB) restoration. Brain pericytes are essential for BBB repair and angiogenesis after stroke, but they also give rise to scar-forming platelet-derived growth factor receptor ß (PDGFR-ß)-expressing cells. However, many of the molecular mechanisms underlying this pericyte response after stroke still remain unknown. Regulator of G-protein signaling 5 (RGS5) has been associated with pericyte detachment from the vascular wall, but whether it regulates pericyte function and vascular stabilization in the chronic phase of stroke is not known. Using RGS5-knockout (KO) mice, we study how loss of RGS5 affects the pericyte response and vascular remodeling in a stroke model at 7 d after ischemia. Loss of RGS5 leads to a shift toward an increase in the number of perivascular pericytes and reduction in the density of parenchymal PDGFR-ß-expressing cells associated with normalized PDGFR-ß activation after stroke. The redistribution of pericytes resulted in higher pericyte coverage, increased vascular density, preservation of vessel lengths, and a significant reduction in vascular leakage in RGS5-KO mice compared with controls. Our study demonstrates RGS5 in pericytes as an important target to enhance vascular remodeling.-Roth, M., Gaceb, A., Enström, A., Padel, T., Genové, G., Özen, I., Paul, G. Regulator of G-protein signaling 5 regulates the shift from perivascular to parenchymal pericytes in the chronic phase after stroke.

Prolonged systemic hyperglycemia does not cause pericyte loss and permeability at the mouse blood-brain barrier.

Diabetes mellitus is associated with cognitive impairment and various central nervous system pathologies such as stroke, vascular dementia, or Alzheimer's disease. The exact pathophysiology of these conditions is poorly understood. Recent reports suggest that hyperglycemia causes cerebral microcirculation pathology and blood-brain barrier (BBB) dysfunction and leakage. The majority of these reports, however, are based on methods including in vitro BBB modeling or streptozotocin-induced diabetes in rodents, opening questions regarding the translation of the in vitro findings to the in vivo situation, and possible direct effects of streptozotocin on the brain vasculature. Here we used a genetic mouse model of hyperglycemia (Ins2AKITA) to address whether prolonged systemic hyperglycemia induces BBB dysfunction and leakage. We applied a variety of methodologies to carefully evaluate BBB function and cellular integrity in vivo, including the quantification and visualization of specific tracers and evaluation of transcriptional and morphological changes in the BBB and its supporting cellular components. These experiments did neither reveal altered BBB permeability nor morphological changes of the brain vasculature in hyperglycemic mice. We conclude that prolonged hyperglycemia does not lead to BBB dysfunction, and thus the cognitive impairment observed in diabetes may have other causes.

Loss of Regulator of G-Protein Signaling 5 Leads to Neurovascular Protection in Stroke.

Background and Purpose- In ischemic stroke, breakdown of the blood-brain barrier (BBB) aggravates brain damage. Pericyte detachment contributes to BBB disruption and neurovascular dysfunction, but little is known about its regulation in stroke. Here, we investigated how loss of RGS5 (regulator of G protein signaling 5) in pericytes affects BBB breakdown in stroke and its consequences. Method- We used RGS5 knockout and control mice and applied a permanent middle cerebral occlusion model. We analyzed pericyte numbers, phenotype, and vessel morphology using immunohistochemistry and confocal microscopy. We investigated BBB breakdown by measuring endothelial coverage, tight junctions, and AQP4 (aquaporin 4) in addition to BBB permeability (fluorescent-conjugated dextran extravasation). Tissue hypoxia was assessed with pimonidazole hydrochloride and neuronal death quantified with the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results- We demonstrate that loss of RGS5 increases pericyte numbers and their endothelial coverage, which is associated with higher capillary density and length, and significantly less BBB damage after stroke. Loss of RGS5 in pericytes results in reduced vascular leakage and preserved tight junctions and AQP4, decreased cerebral hypoxia, and partial neuronal protection in the infarct area. Conclusions- Our findings show that loss of RGS5 affects pericyte-related BBB preservation in stroke and identifies RGS5 as an important target for neurovascular protection.

Hypertension-evoked RhoA activity in vascular smooth muscle cells requires RGS5.

G protein-mediated signaling plays a decisive role in blood pressure regulation and the phenotype of vascular smooth muscle cells (VSMCs); however, the relevance of proteins that restrict G protein activity is not well characterized in this context. Here, we investigated the influence of regulator of G protein signaling 5 (RGS5), an inhibitor of Gαq/11 and Gαi/o activity, on blood pressure and the VSMC phenotype during experimental hypertension. In mice, loss of RGS5 did not affect baseline blood pressure, but prevented hypertension-induced structural remodeling. RGS5-deficient arterial VSMCs did not acquire a synthetic phenotype as evidenced by their inability to decrease the abundance of contractile markers-α-smooth muscle actin and smooth muscle-myosin heavy chain-or to proliferate under these conditions. Mechanistically, hypertensive pressure levels or biomechanical stretch are sufficient to increase the expression of RGS5. Loss of RGS5 severely impairs the activation of RhoA and stress fiber formation. In stretch-exposed VSMCs, RhoA activity was amplified upon inhibition of PKC, which mimics the downstream effects evoked by RGS5-mediated inhibition of Gαq/11 signaling. Collectively, our findings underline that RhoA activation may depend on the restriction of G protein activity and identify RGS5 as a mechanosensitive regulatory protein that is required to promote the synthetic VSMC phenotype as a prerequisite for structural renovation of the arterial wall during hypertension.-Arnold, C., Demirel, E., Feldner, A., Genové, G., Zhang, H., Sticht, C., Wieland, T., Hecker, M., Heximer, S., Korff, T. Hypertension-evoked RhoA activity in vascular smooth muscle cells requires RGS5.

Extracellular retention of PDGF-B directs vascular remodeling in mouse hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) is a lethal condition, and current vasodilator therapy has limited effect. Antiproliferative strategies targeting platelet-derived growth factor (PDGF) receptors, such as imatinib, have generated promising results in animal studies. Imatinib is, however, a nonspecific tyrosine kinase inhibitor and has in clinical studies caused unacceptable adverse events. Further studies are needed on the role of PDGF signaling in PH. Here, mice expressing a variant of PDGF-B with no retention motif ( Pdgfbret/ret), resulting in defective binding to extracellular matrix, were studied. Following 4 wk of hypoxia, right ventricular systolic pressure, right ventricular hypertrophy, and vascular remodeling were examined. Pdgfbret/ret mice did not develop PH, as assessed by hemodynamic parameters. Hypoxia did, however, induce vascular remodeling in Pdgfbret/ret mice; but unlike the situation in controls where the remodeling led to an increased concentric muscularization of arteries, the vascular remodeling in Pdgfbret/ret mice was characterized by a diffuse muscularization, in which cells expressing smooth muscle cell markers were found in the interalveolar septa detached from the normally muscularized intra-acinar vessels. Additionally, fewer NG2-positive perivascular cells were found in Pdgfbret/ret lungs, and mRNA analyses showed significantly increased levels of Il6 following hypoxia, a known promigratory factor for pericytes. No differences in proliferation were detected at 4 wk. This study emphasizes the importance of extracellular matrix-growth factor interactions and adds to previous knowledge of PDGF-B in PH pathobiology. In summary, Pdgfbret/ret mice have unaltered hemodynamic parameters following chronic hypoxia, possibly secondary to a disorganized vascular muscularization.

An Endothelial Gene Signature Score Predicts Poor Outcome in Patients with Endocrine-Treated, Low Genomic Grade Breast Tumors.

PURPOSE: The ability of vascular genes to provide treatment predictive information in breast cancer patients remains unclear. As such, we assessed the expression of genes representative of normal endothelial microvasculature (MV) in relation to treatment-specific patient subgroups. EXPERIMENTAL DESIGN: We used expression data from 993 breast tumors to assess 57 MV genes (summarized to yield an MV score) as well as the genomic grade index (GGI) and PAM50 signatures. MV score was compared with CD31 staining by correlation and gene ontology (GO) analysis, along with clinicopathologic characteristics and PAM50 subtypes. Uni-, multivariate, and/or t-test analyses were performed in all and treatment-specific subgroups, along with a clinical trial cohort of patients with metastatic breast cancer, seven of whom received antiangiogenic therapy. RESULTS: MV score did not correlate with microvessel density (correlation = 0.096), but displayed enrichment for angiogenic GO terms, and was lower in Luminal B tumors. In endocrine-treated patients, a high MV score was associated with decreased risk of metastasis [HR 0.58; 95% confidence interval (CI), 0.38-0.89], even after adjusting for histologic grade, but not GGI or PAM50. Subgroup analysis showed the prognostic strength of the MV score resided in low genomic grade tumors and MV score was significantly increased in metastatic breast tumors after treatment with sunitinib + docetaxel (P = 0.031). CONCLUSIONS: MV score identifies two groups of better and worse survival in low-risk endocrine-treated breast cancer patients. We also show normalization of tumor vasculature on a transcriptional level in response to an angiogenic inhibitor in human breast cancer samples. Clin Cancer Res; 22(10); 2417-26. ©2016 AACR.

Role of Tumor Pericytes in the Recruitment of Myeloid-Derived Suppressor Cells.

BACKGROUND: Pericytes are members of the tumor stroma; however, little is known about their origin, function, or interaction with other tumor components. Emerging evidence suggest that pericytes may regulate leukocyte transmigration. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with powerful inhibitory effects on T-cell-mediated antitumor reactivity. METHODS: We generated subcutaneous tumors in a genetic mouse model of pericyte deficiency (the pdgfb (ret/ret) mouse) and littermate control mice (n = 6-25). Gene expression profiles from 253 breast cancer patients (stage I-III) were evaluated for clinic-pathological parameters and survival using Cox proportional hazard ratios (HRs) and 95% confidence intervals (CIs) based on a two-sided Wald test. RESULTS: We report that pericyte deficiency leads to increased transmigration of Gr1(+)/CD11b(+) cells in experimentally induced tumors. Pericyte deficiency produced defective tumor vasculature, resulting in a more hypoxic microenvironment promoting IL-6 upregulation in the malignant cells. Silencing IL-6 expression in tumor cells attenuated the observed differences in MDSC transmigration. Restoring the pericyte coverage in tumors abrogated the increased MDSC trafficking to pericyte-deficient tumors. MDSC accumulation in tumors led to increases in tumor growth and in circulating malignant cells. Finally, gene expression analysis from human breast cancer patients revealed increased expression of the human MDSC markers CD33 and S100A9 with concomitant decreased expression of pericyte genes and was associated with poor prognosis (HR = 1.88, 95% CI = 1.08 to 3.25, P = .03). CONCLUSIONS: Our data uncovers a novel paracrine interaction between tumor pericytes and inflammatory cells and delineates the cellular events resulting in the recruitment of MDSC to tumors. Furthermore, we propose for the first time a role for tumor pericytes in modulating the expression of immune mediators in malignant cells by promoting a hypoxic microenvironment.

Endogenous brain pericytes are widely activated and contribute to mouse glioma microvasculature.

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. It presents an extremely challenging clinical problem, and treatment very frequently fails due to the infiltrative growth, facilitated by extensive angiogenesis and neovascularization. Pericytes constitute an important part of the GBM microvasculature. The contribution of endogenous brain pericytes to the tumor vasculature in GBM is, however, unclear. In this study, we determine the site of activation and the extent of contribution of endogenous brain pericytes to the GBM vasculature. GL261 mouse glioma was orthotopically implanted in mice expressing green fluorescent protein (GFP) under the pericyte marker regulator of G protein signaling 5 (RGS5). Host pericytes were not only activated within the glioma, but also in cortical areas overlying the tumor, the ipsilateral subventricular zone and within the hemisphere contralateral to the tumor. The host-derived activated pericytes that infiltrated the glioma were mainly localized to the tumor vessel wall. Infiltrating GFP positive pericytes co-expressed the pericyte markers platelet-derived growth factor receptor-ß (PDGFR-ß) and neuron-glial antigen 2. Interestingly, more than half of all PDGFR-ß positive pericytes within the tumor were contributed by the host brain. We did not find any evidence that RGS5 positive pericytes adopt another phenotype within glioma in this paradigm. We conclude that endogenous pericytes become activated in widespread areas of the brain in response to an orthotopic mouse glioma. Host pericytes are recruited into the tumor and constitute a major part of the tumor pericyte population.

Vascular dysfunction and increased metastasis of B16F10 melanomas in Shb deficient mice as compared with their wild type counterparts.

BACKGROUND: Shb is a signaling protein downstream of vascular endothelial growth factor receptor-2 and Shb deficiency has been found to restrict tumor angiogenesis. The present study was performed in order to assess metastasis in Shb deficiency using B16F10 melanoma cells. METHODS: B16F10 melanoma cells were inoculated subcutaneously on wild type or Shb +/- mice. Primary tumors were resected and lung metastasis determined after tumor relapse. Lung metastasis was also assessed after bone marrow transplantation of wild type bone marrow to Shb +/- recipients and Shb +/- bone marrow to wild type recipients. Primary tumors were subject to immunofluorescence staining for CD31, VE-cadherin, desmin and CD8, RNA isolation and isolation of vascular fragments for further RNA isolation. RNA was used for real-time RT-PCR and microarray analysis. RESULTS: Numbers of lung metastases were increased in Shb +/- or -/- mice and this coincided with reduced pericyte coverage and increased vascular permeability. Gene expression profiling of vascular fragments isolated from primary tumors and total tumor RNA revealed decreased expression of different markers for cytotoxic T cells in tumors grown on Shb +/- mice, suggesting that vascular aberrations caused altered immune responses. CONCLUSIONS: It is concluded that a unique combinatorial response of increased vascular permeability and reduced recruitment of cytotoxic CD8+ cells occurs as a consequence of Shb deficiency in B16F10 melanomas. These changes may promote tumor cell intravasation and metastasis.

Animal models of diabetic macrovascular complications: key players in the development of new therapeutic approaches.

Diabetes mellitus is a lifelong, incapacitating metabolic disease associated with chronic macrovascular complications (coronary heart disease, stroke, and peripheral vascular disease) and microvascular disorders leading to damage of the kidneys (nephropathy) and eyes (retinopathy). Based on the current trends, the rising prevalence of diabetes worldwide will lead to increased cardiovascular morbidity and mortality. Therefore, novel means to prevent and treat these complications are needed. Under the auspices of the IMI (Innovative Medicines Initiative), the SUMMIT (SUrrogate markers for Micro- and Macrovascular hard end points for Innovative diabetes Tools) consortium is working on the development of novel animal models that better replicate vascular complications of diabetes and on the characterization of the available models. In the past years, with the high level of genomic information available and more advanced molecular tools, a very large number of models has been created. Selecting the right model for a specific study is not a trivial task and will have an impact on the study results and their interpretation. This review gathers information on the available experimental animal models of diabetic macrovascular complications and evaluates their pros and cons for research purposes as well as for drug development.

Increased flux of the plant sterols campesterol and sitosterol across a disrupted blood brain barrier.

The intact blood-brain barrier in mammalians prevents exchange of cholesterol loaden particles between periphery and brain and thus nearly all cholesterol in this organ originates from de novo synthesis. Dietary cholesterol homologues from plants, campesterol and sitosterol, are known to get enriched to some extent in the mammalian brain. We recently showed that Pdgfb(ret)(/)(ret) mice, with a pericyte deficiency and a leaking blood-brain barrier phenotype, have significantly higher levels of plant sterols in the brain compared to their heterozygous Pdgfb(ret)(/)(+) controls keeping the integrity of the blood-brain barrier (BBB). In order to further study the protective functionality of the BBB we synthesized a mixture of [(2)H6]campesterol/sitosterol and fed it for 10-40days to genetically different types of animals. There was a significant enrichment of both deuterium stable isotope labeled plant sterols in the brain of both strains of mice, however, with a lower enrichment in the controls. As expected, the percentage and absolute enrichment was higher for [(2)H6]campesterol than for the more lipophilic [(2)H6]sitosterol. The results confirm that a leaking BBB causes increased flux of plant sterols into the brain. The significant flux of the labeled plant sterols into the brain of the control mice illustrates that the presence of an alkyl group in the 24-position of the steroid side chain markedly increases the ability of cholesterol to pass an intact BBB. We discuss the possibility that there is a specific transport mechanism involved in the flux of alkylated cholesterol species across the BBB.