Correction to: The Microvascular Pericyte: Approaches to Isolation, Characterization, and Cultivation.

The author has spotted an error on page 61, in the middle of the second paragraph from bottom. The content has been revised and the corrected version is as follows.

The Microvascular Pericyte: Approaches to Isolation, Characterization, and Cultivation.

The microvascular pericyte was identified in 1873 by the French scientist Charles Benjamin Rouget and originally called the Rouget cell (Rouget.Sciences 88:916-8, 1879). However, it was not until the early 1900s that Rouget's work was confirmed, and the Rouget cell renamed the pericyte by virtue of its peri-endothelial location (Dore. Brit J Dermatol 35:398-404, 1923; Zimmermann. Z Anat Entwicklungsgesch 68:3-109, 1923). Over the years a large number of publications have emerged, but the pericyte has remained a truly enigmatic cell. This is due, in part, by the paucity of easy and reliable methods to isolate and characterize the cell as well as its heterogeneity and pluripotent characteristics. However, more recent advances in molecular genetics and development of novel cell isolation and imaging techniques have enable scientists to more readily define pericyte function. This chapter will discuss general approaches to the isolation, characterization, and propagation of primary pericytes in the establishment of cell lines. We will attempt to dispel misinterpretations about the pericyte that cloud the literature.

Endogenous adaptation to low oxygen modulates T-cell regulatory pathways in EAE.

BACKGROUND: In the brain, chronic inflammatory activity may lead to compromised delivery of oxygen and glucose suggesting that therapeutic approaches aimed at restoring metabolic balance may be useful. In vivo exposure to chronic mild normobaric hypoxia (10 % oxygen) leads to a number of endogenous adaptations that includes vascular remodeling (angioplasticity). Angioplasticity promotes tissue survival. We have previously shown that induction of adaptive angioplasticity modulates the disease pattern in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). In the present study, we define mechanisms by which adaptation to low oxygen functionally ameliorates the signs and symptoms of EAE and for the first time show that tissue hypoxia may fundamentally alter neurodegenerative disease. METHODS: C57BL/6 mice were immunized with MOG, and some of them were kept in the hypoxia chambers (day 0) and exposed to 10 % oxygen for 3 weeks, while the others were kept at normoxic environment. Sham-immunized controls were included in both hypoxic and normoxic groups. Animals were sacrificed at pre-clinical and peak disease periods for tissue collection and analysis. RESULTS: Exposure to mild hypoxia decreased histological evidence of inflammation. Decreased numbers of cluster of differentiation (CD)4+ T cells were found in the hypoxic spinal cords associated with a delayed Th17-specific cytokine response. Hypoxia-induced changes did not alter the sensitization of peripheral T cells to the MOG peptide. Exposure to mild hypoxia induced significant increases in anti-inflammatory IL-10 levels and an increase in the number of spinal cord CD25+FoxP3+ T-regulatory cells. CONCLUSIONS: Acclimatization to mild hypoxia incites a number of endogenous adaptations that induces an anti-inflammatory milieu. Further understanding of these mechanisms system may pinpoint possible new therapeutic targets to treat neurodegenerative disease.

Hypoxia-Induced Let-7d Has a Role in Pericyte Differentiation.

The microvascular pericyte is an important regulatory cell that maintains tissue homeostasis. One of the mechanisms by which pericytes maintain tissue homeostasis is through the induction of endogenous adaptative changes to stress signals. These adaptations include migration, differentiation and induction of angiogenesis. We have investigated pericyte responses to hypoxic stress (1 % O2) and have reported that pericytes adapt to hypoxia, in part, through changes in endogenous and released microRNAs (miRNAs). Of those miRNAs, Let-7d plays an important role. We exposed pericytes to hypoxia with and without basic fibroblast growth factor (bFGF) in stem cell medium. The expression of Let-7d in pericyte-derived neurospheres was determined. Evidence of differentiation was determined by immunocytochemistry. Hypoxia enhanced pericyte spheres were positive for Let-7d. The transcription factor Sox2, a marker of cell differentiation, was also induced in pericytic spheres. Taken together, our results suggest that pericyte expression of Let-7d in response to hypoxia and bFGF is involved in pericyte differentiation. Thus, for the first time, we propose a pathway for induction of pericyte differentiation. Modulation of this pathway in pericytes may be an important target in tissue repair.

Chronic cerebrospinal venous insufficiency and multiple sclerosis.

A chronic state of impaired venous drainage from the central nervous system, termed chronic cerebrospinal venous insufficiency (CCSVI), is claimed to be a pathologic phenomenon exclusively seen in multiple sclerosis (MS). This has invigorated the causal debate of MS and generated immense interest in the patient and scientific communities. A potential shift in the treatment paradigm of MS involving endovascular balloon angioplasty or venous stent placement has been proposed as well as conducted in small patient series. In some cases, it may have resulted in serious injury. In this Point of View, we discuss the recent investigations that led to the description of CCSVI as well as the conceptual and technical shortcomings that challenge the potential relationship of this phenomenon to MS. The need for conducting carefully designed and rigorously controlled studies to investigate CCVSI has been recognized by the scientific bodies engaged in MS research. Several scientific endeavors examining the presence of CCSVI in MS are being undertaken. At present, invasive and potentially dangerous endovascular procedures as therapy for patients with MS should be discouraged until such studies have been completed, analyzed, and debated in the scientific arena.

Immortalized CNS pericytes are quiescent smooth muscle actin-negative and pluripotent.

Despite their identification more than 100 years ago by the French scientist Charles-Marie Benjamin Rouget, microvascular pericytes have proven difficult to functionally characterize, due in part to their relatively low numbers and the lack of specific cell markers. However, recent progress is beginning to shed light on the diverse biological functions of these cells. Pericytes are thought to be involved in regulating vascular homeostasis and hemostasis as well as serving as a local source of adult stem cells. To further define the properties of these intriguing cells, we have isolated pericytes from transgenic mice (Immortomouse®) harboring a temperature-sensitive mutant of the SV40 virus target T-gene. This Immortopericyte (IMP) conditional cell line is stable for long periods of time and, at 33°C in the presence of interferon gamma, does not differentiate. Under these conditions IMPs are alpha muscle actin-negative and exhibit a pluripotent phenotype, but can be induced to differentiate along both mesenchymal and neuronal lineages at 37°C. Alternatively, differentiation of wild type pericytes and IMPs can be induced directly from capillaries in culture. Finally, the addition of endothelial cells to purified IMP cultures augments their rate of self-renewal and differentiation, possibly in a cell-to-cell contact dependent manner.

Chronic mild hypoxia ameliorates chronic inflammatory activity in myelin oligodendrocyte glycoprotein (MOG) peptide induced experimental autoimmune encephalomyelitis (EAE).

While the pathologic events associated with multiple sclerosis (MS), diffuse axonal injury, cognitive damage, and white matter plaques, have been known for some time, the etiology of MS is still unknown and therapeutic efforts are somewhat disappointing. This may be due to a lack of fundamental knowledge on how to buffer the brain from secondary injury following immune attack. Maintenance of central nervous system (CNS) homeostasis is a complex set of regulatory adjustments by the neurovascular unit that includes induction of adaptive angiogenesis. Although aspects of adaptive angiogenesis are induced in MS and experimental autoimmune encephalomyelitis (EAE), vascular remodeling is ineffective and the balance between metabolic need and oxygen (O(2)) and glucose availability is disrupted.We hypothesized that restoration of metabolic homeostasis in the CNS would ameliorate tissue damage and promote repair in myelin oligodendrocyte glycoprotein(MOG)-induced EAE in mice. Exposure of animals to chronic mild hypoxia (10% O(2)) increased vascular density and significantly delayed onset and severity of clinical EAE. When animals were exposed to hypoxia after the onset of clinical symptoms, the severity of chronic inflammatory disease was reduced or even inhibited. In addition, spinal cord pathology was decreased.While the mechanism of protection is unclear, results suggest that hypoxia has therapeutic potential in EAE.

In the hypoxic central nervous system, endothelial cell proliferation is followed by astrocyte activation, proliferation, and increased expression of the alpha 6 beta 4 integrin and dystroglycan.

Cerebral hypoxia induces a profound angiogenic response in the central nervous system (CNS). Using a mouse model of chronic cerebral hypoxia, we previously demonstrated that angiogenic vessels in the hypoxic CNS show marked upregulation of the extracellular matrix (ECM) protein fibronectin, along with increased expression of its major receptor, alpha 5 beta 1 integrin on brain endothelial cells (BEC). As cerebral hypoxia also leads to glial activation, the aim of the current study was to define the temporal relationship between BEC responses and glial cell activation in this model of cerebral hypoxia. This revealed that BEC fibronectin/alpha 5 beta 1 integrin expression and proliferation both reached maximal level after 4-day hypoxia. Interestingly, up to 4-day hypoxia, all dividing cells were BEC, but at later time-points proliferating astrocytes were also observed. GFAP staining revealed that hypoxia induced marked astrocyte activation that reached maximal level between 7- and 14-day hypoxia. As newly formed cerebral capillaries require ensheathment by astrocyte end-feet to acquire mature brain endothelium characteristics, we next examined how expression of astrocyte end-feet adhesion molecules is regulated by hypoxia. This showed that the astrocyte adhesion receptors alpha 6 beta 4 integrin and dystroglycan were both markedly upregulated, with a time-course that closely resembled astrocyte activation. Taken together, this evidence shows that cerebral hypoxia promotes first an endothelial response, in which fibronectin promotes BEC proliferation. This is then followed by an astrocyte response, involving astrocyte activation, proliferation, and reorganization of astrocyte end-feet, which correlates with increased expression of astrocyte end-feet adhesion molecules.

Increased expression of fibronectin and the alpha 5 beta 1 integrin in angiogenic cerebral blood vessels of mice subject to hypobaric hypoxia.

The extracellular matrix (ECM) is an important regulator of angiogenesis and vascular remodeling. We showed previously that angiogenic capillaries in the developing CNS express high levels of fibronectin and its receptor alpha5beta1 integrin, and that this expression is developmentally downregulated. As cerebral hypoxia leads to an angiogenic response, we sought to determine whether angiogenic vessels in the adult CNS re-express fibronectin and the alpha5beta1 integrin. Ten-week old mice were subject to hypobaric hypoxia for 0, 4, 7 and 14 days, and fibronectin/integrin expression examined. Fibronectin and the alpha5 integrin subunit were strongly upregulated on capillaries in the hypoxic CNS, with the effect maximal at the earliest time point examined (4 days). Immunofluorescent studies demonstrated that the alpha5 integrin was expressed by angiogenic endothelial cells. In light of the defined angiogenic role for fibronectin in other systems, this work suggests that induction of fibronectin-alpha5beta1 integrin expression may be an important molecular switch driving angiogenesis in the hypoxic CNS.

Physiologic angiodynamics in the brain.

Hypoxic acclimatization includes increased brain capillary density. Adaptive angiogenesis, which occurs over a 3-week period, is mediated by upregulation of vascular endothelial growth factor induced by hypoxia-inducible factor-1 in concert with the capillary remodeling molecule angiopoietin-2, which is upregulated through cyclooxygenase-2 production of prostaglandin E2. The process is apparently orchestrated by pericytes, which regulate the microvascular milieu and coordinate the interactions within the neurovascular unit. The return to normoxia is accompanied by microvascular regression and decreasing numbers of capillaries to prehypoxic densities. Regression is the result of endothelial cell apoptosis, suggesting the existence of physiologic mechanisms for adjusting capillary density to balance oxygen availability and oxygen consumption. The capacity for adaptation is diminished in older rats because of the attenuation of the hypoxia-inducible factor-1 response.

Differential expression of capillary VEGF isoforms following traumatic brain injury.

OBJECTIVES: While it is known that angiogenesis occurs after trauma, we sought to characterize the expression of vascular endothelial growth factor (VEGF) subtypes, vascular endothelial growth factor receptor 2 (VEGFR2) and angiopoietin within capillaries of animals subjected to traumatic brain injury (TBI). Further, we sought to characterize pericyte cell death in isolated capillaries. METHODS: We used Marmarou's acceleration impact model to induce head trauma and measured VEGF, VEGFR2 and angiopoietin levels in isolated capillaries. TUNEL was used to determine pericyte cell death. RESULTS: The VEGF response was restricted to the VEGF120 isoform. No increase in transcripts for VEGF164 and VEGF188 was observed. VEGFR2 was marginally increased and angiopoietin was increased. A subset of pericytes were TUNEL-positive. DISCUSSION: These results show a distinct expression pattern of angiogenic factors following injury and suggest that pericyte involvement in adaptive angiogenesis may be altered following TBI.

CNS microvascular pericytes exhibit multipotential stem cell activity.

It has been suggested that a vascular-like cell has multipotent regenerative and mesenchymal lineage relationships. The identity of this stem/progenitor cell has remained elusive. We report here that adult central nervous system (CNS) capillaries contain a distinct population of microvascular cells, the pericyte that are nestin/NG2 positive and in response to basic fibroblast growth factor (bFGF) differentiate into cells of neural lineage. In their microvascular location, pericytes express nestin and NG2 proteoglycan. In serum containing media primary (0 to 7 day old) CNS pericytes are nestin positive, NG2 positive, alpha smooth muscle actin (alphaSMA) positive, and do not bind the endothelial cell specific griffonia symplicifolia agglutinin (GSA). In serum containing media, pericytes do not undergo neurogenesis but are induced to express alphaSMA. In bFGF containing media without serum, CNS pericytes form small clusters and multicellular spheres. Differentiated spheres expressed neuronal and glial cell markers. After disruption and serial dilution, differentiated spheres were capable of self-renewal. When differentiated spheres were disrupted and cultured in the presence of serum, multiple adherent cell populations were identified by dual and triple immunocytochemistry. Cells expressing markers characteristic of pericytes, neurons, and glial cells were generated. Many of the cells exhibited dual expression of differentiation markers. With prolonged culture fully differentiated cells of neural lineage were present. Results indicate that adult CNS microvascular pericytes have neural stem cell capability.

New frontiers in translational research in neuro-oncology and the blood-brain barrier: report of the tenth annual Blood-Brain Barrier Disruption Consortium Meeting.

The blood-brain barrier (BBB) presents a major obstacle to the treatment of malignant brain tumors and other central nervous system (CNS) diseases. For this reason, a meeting partially funded by an NIH R13 grant was convened to discuss recent advances and future directions in translational research in neuro-oncology and the BBB. Cell biology and transport across the BBB, delivery of agents to the CNS, neuroimaging, angiogenesis, immunotherapy, and gene therapy, as well as glioma, primary CNS lymphoma, and metastases to the CNS were discussed. Transport across the BBB relates to the neurovascular unit, which consists not only of endothelial cells but also of pericyte, glia, and neuronal elements.

Cytokine regulation of E-selectin in rat CNS microvascular endothelial cells: differential response of CNS and non-CNS vessels.

We have compared the induced expression of E-selectin in primary cultures of rat brain microvascular endothelial cells (EC), pericytes and in non-CNS microvascular endothelium stimulated with the cytokines, IL-1beta (20 ng/ml), and tumor necrosis factor (TNF)-alpha (75 ng/ml). Expression was studied at both the protein and mRNA levels. Fluorescence in-situ hybridization (FISH) was used to examine de novo synthesis of E-selectin mRNA. Laser cytometric analysis was used as a novel approach to the quantitaion of FISH. In-situ hybridization was performed using two PCR-generated probes. The first probe (517 bp) spanned the lectin and epidermal growth factor (EGF)-like domain. The second probe (562 bp) spanned the CR3, 4, and 6 domains. E-selectin-specific mRNA was localized to the perinuclear regions of the EC. Both cytokines, IL-1beta and TNF-alpha significantly increased E-selectin gene expression in CNS EC but not pericytes. IL-1beta induced higher E-selectin mRNA levels than TNF-alpha. The maximum number of mRNA-positive cells was observed after stimulation for 4--6 h. Surface protein expression was sustained for up to 48 h following addition of cytokine. This was in contrast to the transient expression in non-CNS EC indicating that pure primary CNS EC display slightly different kinetics of E-selectin expression than non-CNS EC.

Pericytes and adaptive angioplasticity: the role of tumor necrosis factor-like weak inducer of apoptosis (TWEAK).

The TNF superfamily member TWEAK has emerged as a pleiotropic cytokine that regulates many cellular functions that include immune/inflammatory activity, angiogenesis, cell proliferation, and fate. TWEAK through its inducible receptor, FGF-inducible molecule 14 (Fn14), can induce both beneficial and deleterious activity that has a profound effect on cell survival. Thus it is highly likely that TWEAK and Fn14 expressed by cells of the neurovascular unit help regulate and maintain vascular and tissue homeostasis. In this chapter we discuss the expression of TWEAK and Fn14 signaling in the cerebral microvascular pericyte. Pericytes are a highly enigmatic population of microvascular cells that are important in regulatory pathways that modulate physiological angiogenesis in response to chronic mild hypoxic stress. A brief introduction will identify the microvascular pericyte. A more detailed discussion of pericyte TWEAK signaling during adaptive angioplasticity will follow.

Induction of vascular remodeling: a novel therapeutic approach in EAE.

While the pathologic events associated with multiple sclerosis (MS), diffuse axonal injury, cognitive damage, and white matter plaques, have been known for some time, their etiology is unknown and therapeutic efforts are still somewhat disappointing. This may be due to a lack of fundamental knowledge on how to maintain tissue homeostasis and buffer the brain from secondary injury. Maintenance of homeostasis in the brain is the result of regulatory adjustments by cellular constituents of the neurovascular unit (pericytes, endothelial cells, astrocytes, and neurons) that include induction of adaptive vascular remodeling. Results from our laboratory and others suggest that aspects of stress induced adaptation are seen in MS and in the murine model of experimental autoimmune encephalomyelitis (EAE), vascular remodeling is ineffective and biometabolic balance is disrupted. In murine white matter, capillary density is 1/2 that observed in gray matter thus disruption of vascular homeostasis will have a profound impact on tissue integrity. We therefore hypothesized that restoration of microvascular angiodynamics would augment tissue plasticity mitigating the extent of secondary injury and sparing cognitive decline in patients with MS. To test this hypothesis, we have performed preclinical studies and characterized changes in angiodynamics in myelin oligodendrocyte glycoprotein (MOG) peptide (35-55)-induced EAE in C57BL/6 mice with or without concomitant exposure to chronic mild low oxygen. We have reported that exposure to chronic mild low oxygen ameliorated clinical disease in EAE. While the mechanisms of protection are unclear, results suggest that normobaric hypoxia stabilizes the stress response, promotes physiological angiogenesis, and is neuroprotective.

Hypoxia alters MicroRNA expression in rat cortical pericytes.

Microvascular adaptation to metabolic stress is important in the maintenance of tissue homeostasis. Nowhere is this more important than in the central nervous system (CNS) where the cellular constituents of the neurovascularture including endothelial cells, pericytes and some astroglia must make fine-tuned autoregulatory modulations that maintain the delicate balance between oxygen availability and metabolic demand. miRNAs have been reported to play an important regulatory role in many cellular functions including cell differentiation, growth and proliferation, lineage determination, and metabolism. In this study, we investigated the possible role of miRNAs in the CNS capillary pericyte response to hypoxic stress. Micro-array analysis was used to examine the expression of 388 rat miRNAs in primary rat cortical pericytes with and without exposure to low oxygen (1%) after 24 or 48 hr. Pericytes subjected to hypoxia showed 27 miRNAs that were higher than control and 31 that were lower. Validation and quantification was performed by Real Time RT-PCR on pericytes subjected to 2 hr, 24 hr, or 48 hr of hypoxia. Hypoxia induced changes included physiological pathways governing the stress response, angiogenesis, migration and cell cycle regulation. miRNAs associated with HIF-1α (miR-322[1], miR-199a [2]), TGF-ß1 (miR-140[3], miR-145[4], miR-376b-3p[5]) and VEGF (miR-126a[6], miR-297[7], miR-16[8], miR-17-5p[9]) were differentially regulated. Systematic and integrative analysis of possible gene targets analyzed by DAVID bioinformatics resource (http://david.abcc.ncifcrf.gov) and MetaSearch 2.0 (GeneGo) for some of these miRNAs was conducted to determine possible gene targets and pathways that may be affected by the post-transcriptional changes after hypoxic insult.

Pericyte coculture models to study astrocyte, pericyte, and endothelial cell interactions.

The microvascular pericyte is an integral component of the blood-brain barrier and the neurovascular unit. Most model systems that have been developed to study the functional parameters of these systems have not incorporated the pericyte. In this chapter, we consider pericyte coculture and triple culture systems and detail the methodology, suggestions, and problems with isolation of these unique cells. We also present data to show that triple cultures are ideal to study the role of the CNS pericyte in CNS angiogenesis.

Enhanced cerebral but not peripheral angiogenesis in the Goto-Kakizaki model of type 2 diabetes involves VEGF and peroxynitrite signaling.

We previously reported enhanced cerebrovascular remodeling and arteriogenesis in experimental type 2 diabetes. This study tested the hypotheses that 1) cerebral but not peripheral angiogenesis is increased in a spatial manner and 2) peroxynitrite orchestrates vascular endothelial growth factor (VEGF)-mediated brain angiogenesis in diabetes. Stereology of brain, eye, and skeletal muscle microvasculature was evaluated in control and diabetic rats using three-dimensional images. Migration and tube formation properties of brain microvascular endothelial cells (BMECs) were analyzed as markers of angiogenesis. Vascular density, volume, and surface area were progressively increased from rostral to caudal sections in both the cerebral cortex and striatum in diabetic rats. Unperfused new vessels were more prominent and the pericyte-to-endothelial cell ratio was decreased in diabetes. Vascularization was greater in the retina but lower in the peripheral circulation. VEGF and nitrotyrosine levels were higher in cerebral microvessels of diabetic animals. Migratory and tube formation properties were enhanced in BMECs from diabetic rats, which also expressed high levels of basal VEGF, nitrotyrosine, and membrane-type (MT1) matrix metalloprotease (MMP). VEGF-neutralizing antibody and inhibitors of peroxynitrite, src kinase, or MMP blocked the migration. Diabetes increases and spatially regulates cerebral neovascularization. Increased VEGF-dependent angiogenic function in BMECs is mediated by peroxynitrite and involves c-src and MT1-MMP activation.

Morphology and properties of pericytes.

Pericytes were described in 1873 by the French scientist Charles-Marie Benjamin Rouget and were originally called Rouget cells. The Rouget cell was renamed some years later due to its anatomical location abluminal to the endothelial cell (EC) and luminal to parenchymal cells. In the brain, pericytes are located in precapillary arterioles, capillaries and postcapillary venules. They deposit elements of the basal lamina and are totally surrounded by this vascular component. Pericytes are important cellular constituents of the blood-brain barrier (BBB) and actively communicate with other cells of the neurovascular unit such as ECs, astrocytes, and neurons. Pericytes are local regulatory cells that are important for the maintenance of homeostasis and hemostasis, and are a source of adult pluripotent stem cells. Further understanding of the role played by this intriguing cell may lead to novel targeted therapies for neurovascular diseases.