Cell type-selective secretome profiling in vivo.

Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

Imaging and optogenetic modulation of vascular mural cells in the live brain.

Mural cells (smooth muscle cells and pericytes) are integral components of brain blood vessels that play important roles in vascular formation, blood-brain barrier maintenance, and regulation of regional cerebral blood flow (rCBF). These cells are implicated in conditions ranging from developmental vascular disorders to age-related neurodegenerative diseases. Here we present complementary tools for cell labeling with transgenic mice and organic dyes that allow high-resolution intravital imaging of the different mural cell subtypes. We also provide detailed methodologies for imaging of spontaneous and neural activity-evoked calcium transients in mural cells. In addition, we describe strategies for single- and two-photon optogenetics that allow manipulation of the activity of individual and small clusters of mural cells. Together with measurements of diameter and flow in individual brain microvessels, calcium imaging and optogenetics allow the investigation of pericyte and smooth muscle cell physiology and their role in regulating rCBF. We also demonstrate the utility of these tools to investigate mural cells in the context of Alzheimer's disease and cerebral ischemia mouse models. Thus, these methods can be used to reveal the functional and structural heterogeneity of mural cells in vivo, and allow detailed cellular studies of the normal function and pathophysiology of mural cells in a variety of disease models. The implementation of this protocol can take from several hours to days depending on the intended applications.

A pump-free tricellular blood-brain barrier on-a-chip model to understand barrier property and evaluate drug response.

Disruption of the blood-brain barrier (BBB) leads to various neurovascular diseases. Development of therapeutics required to cross the BBB is difficult due to a lack of relevant in vitro models. We have developed a three-dimensional (3D) microfluidic BBB chip (BBBC) to study cell interactions in the brain microvasculature and to test drug candidates of neurovascular diseases. We isolated primary brain microvascular endothelial cells (ECs), pericytes, and astrocytes from neonatal rats and cocultured them in the BBBC. To mimic the 3D in vivo BBB structure, we used type I collagen hydrogel to pattern the microchannel via viscous finger patterning technique to create a matrix. ECs, astrocytes, and pericytes were cocultured in the collagen matrix. The fluid flow in the BBBC was controlled by a pump-free strategy utilizing gravity as driving force and resistance in a paper-based flow resistor. The primary cells cultured in the BBBC expressed high levels of junction proteins and formed a tight endothelial barrier layer. Addition of tumor necrosis factor alpha to recapitulate neuroinflammatory conditions compromised the BBB functionality. To mitigate the neuroinflammatory stimulus, we treated the BBB model with the glucocorticoid drug dexamethasone, and observed protection of the BBB. This BBBC represents a new simple, cost-effective, and scalable in vitro platform for validating therapeutic drugs targeting neuroinflammatory conditions.

Isolation, cultivation, and characterization of primary bovine cochlear pericytes: A new in vitro model of stria vascularis.

The study of strial pericytes has gained great interest as they are pivotal for the physiology of stria vascularis. To provide an easily accessible in vitro model, here we described a growth medium-based approach to obtain and cultivate primary bovine cochlear pericytes (BCP) from the stria vascularis of explanted bovine cochleae. We obtained high-quality pericytes in 8-10 days with a > 90% purity after the second passage. Immunocytochemical analysis showed a homogeneous population of cells expressing typical pericyte markers, such as neural/glial antigen 2 (NG2), platelet-derived growth factor receptorß (PDGFRß), α-smooth muscle actin (α-SMA), and negative for the endothelial marker von Willebrand factor. When challenged with tumor necrosis factor or lipopolysaccharide, BCP changed their shape, similarly to human retinal pericytes (HRPC). The sensitivity of BCP to ototoxic drugs was evaluated by challenging with cisplatin or gentamicin for 48 hr. Compared to human retinal endothelial cells and HRPC, cell viability of BCP was significantly lower ( p < 0.05) after the treatment with gentamicin or cisplatin. These data indicate that our protocol provides a simple and reliable method to obtain highly pure strial BCP. Furthermore, BCP are suitable to assess the safety profile of molecules which supposedly exert ototoxic activity, and may represent a valid alternative to in vivo tests.

[Pathomechanisms of pericyte-myofibroblast transition in kidney and interventional effects of Chinese herbal medicine].

In the kidney, pericyte is the major source of myofibroblast (MyoF) in renal interstitium. It is reported that pericyte-myofibroblast transition（PMT）is one of the important pathomechanisms of renal interstitial fibrosis（RIF）. Among them, the main reasons for promoting RIF formation include pericyte recruitment, activation and isolation, as well as the lack of pericyte-derived erythropoietin. During the PMT startup process, pericyte activation and its separation from microvessels are controlled by multiple signal transduction pathways, such as transforming growth factor-ß（TGF-ß）pathway, vascular endothelial growth factor receptor (VEGFR) pathway and platelet derived growth factor receptor (PDGFR) pathway;Blocking of these signaling pathways can not only inhibit PMT, but also suppress renal capillaries reduction and further alleviate RIF. In clinic, many traditional Chinese medicine compound prescriptions, single traditional Chinese herbal medicine (CHM) and their extracts have the clear effects in alleviating RIF, and some of their intervention actions may be related to pericyte and its PMT. Therefore, the studies on PMT and its drug intervention will become the main development direction in the research field of anti-organ fibrosis by CHM.

[Recent advances on pericytes in microvascular dysfunction and traditional Chinese medicine prevention].

Pericytesis a kind of widespread vascular mural cells embedded within the vascular basement membrane of blood microvessels, constituting the barrier of capillaries and tissue spaces together with endothelial cells. Pericytes communicate with microvascular endothelial cells through cell connections or paracrine signals, playing an important role in important physiological processes such as blood flow, vascular permeability and vascular formation. Pericytes dysfunction may participate in some microvascular dysfunction, and also mediate pathological repair process, therefore pericytes attracted more and more attention. Traditional Chinese medicine suggests that microvascular dysfunction belongs to the collaterals disease; Qi stagnation and blood stasis in collaterals result in function imbalance of internal organs. Traditional Chinese medicine (TCM) has shown effects on pericytes in microvascular dysfunction, for example qi reinforcing blood-circulation activating medicines can reduce the damage of retinal pericytes in diabetic retinopathy. However, there are some limitations of research fields, inaccuracy of research techniques and methods, and lack of mechanism elaboration depth in the study of microvascular lesion pericytes. This paper reviewed the biological characteristics of pericytes and pericytes in microvascular dysfunction, as well as the intervention study of TCM on pericytes. The article aims to provide reference for the research of pericytes in microvascular dysfunction and the TCM study on pericytes.

Environmental Enrichment Induces Pericyte and IgA-Dependent Wound Repair and Lifespan Extension in a Colon Tumor Model.

Environmental enrichment (EE) replicates mind-body therapy by providing complex housing to laboratory animals to improve their activity levels, behavior, and social interactions. Using a Tcf4Het/+ApcMin/+-mediated model of colon tumorigenesis, we found that EE vastly improved the survival of tumor-bearing animals, with differential effect on tumor load in male compared to female animals. Analysis of Tcf4Het/+ApcMin/+ males showed drastically reduced expression of circulating inflammatory cytokines and induced nuclear hormone receptor (NHR) signaling, both of which are common in the wound repair process. Interestingly, EE provoked tumor wound repair resolution through revascularization, plasma cell recruitment and IgA secretion, replacement of glandular tumor structures with pericytes in a process reminiscent of scarring, and normalization of microbiota. These EE-dependent changes likely underlie the profound improvement in survival of colon-tumor-bearing Tcf4Het/+ApcMin/+ males. Our studies highlight the exciting promise of EE in the design of future therapeutic strategies for colon cancer patients.

Inhibitory effect of Angelica gigas on cold­induced RhoA activation in vascular cells.

The herbal extract Angelica gigas (AG) has been applied as a vasodilating agent for patients suffering from vascular diseases for many years; however, the underlying mechanism has not been fully elucidated. The present study hypothesized that the anti­vasoconstrictive effect of AG may be effective in the treatment of abnormal cold­mediated vasospasms that occur in Raynaud's phenomenon (RP). The effect of AG on the activity of ras homolog gene family member A (RhoA) was investigated in cold­exposed vascular cells. Vascular cells were pretreated to AG, followed by a warm (37˚C) or cold (25˚C) incubation for 30 min and investigated with western blotting, ELISA and confocal microscopy. Cold treatment induced the activation of RhoA in pericytes and vascular endothelial cells, however this was reduced by treatment with AG. Furthermore, AG treatment reduced the endothelin­1 (ET­1)­mediated RhoA activation in pericytes; however, cold­induced ET­1 production by vascular endothelial cells was not affected by treatment with AG. In addition, AG treatment suppressed the formation of stress fibers and focal adhesion complexes, and the cold­induced phosphorylation of focal adhesion kinase, proto­oncogene tyrosine­protein kinase Src and extracellular signal­related kinase. Therefore, AG treatment demonstrated an ability to reduce cold­induced RhoA activation in pericytes and vascular endothelial cells, and attenuated ET­1­mediated RhoA activation in pericytes. In conclusion, the present study indicated that AG may be useful for the treatment of RP.

Brain pericyte-derived soluble factors enhance insulin sensitivity in GT1-7 hypothalamic neurons.

Insulin signaling in the hypothalamus plays an important role in food intake and glucose homeostasis. Hypothalamic neuronal functions are modulated by glial cells; these form an extensive network connecting the neurons and cerebral vasculature, known as the neurovascular unit (NVU). Brain pericytes are periendothelial accessory structures of the blood-brain barrier and integral members of the NVU. However, the interaction between pericytes and neurons is largely unexplored. Here, we investigate whether brain pericytes could affect hypothalamic neuronal insulin signaling. Our immunohistochemical observations demonstrated the existence of pericytes in the mouse hypothalamus, exhibiting immunoreactivity of platelet-derived growth factor receptor ß (a pericyte marker), and laminin, a basal lamina marker. We then exposed a murine hypothalamic neuronal cell line, GT1-7, to conditioned medium obtained from primary cultures of rat brain pericytes. Pericyte-conditioned medium (PCM), but not astrocyte- or aortic smooth muscle cell-conditioned medium, increased the insulin-stimulated phosphorylation of Akt in GT1-7 cells in a concentration-dependent manner. PCM also enhanced insulin-stimulated tyrosine phosphorylation of insulin receptor ß without changing its expression or localization in cytosolic or plasma membrane fractions. These results suggest that pericytes, rather than astrocytes, increase insulin sensitivity in hypothalamic neurons by releasing soluble factors under physiological conditions in the NVU.

Changes in pericytic expression of NG2 and PDGFRB and vascular permeability in the sensory circumventricular organs of adult mouse by osmotic stimulation.

The blood-brain barrier (BBB) is a barrier that prevents free access of blood-derived substances to the brain through the tight junctions and maintains a specialized brain environment. Circumventricular organs (CVOs) lack the typical BBB. The fenestrated vasculature of the sensory CVOs, including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows parenchyma cells to sense a variety of blood-derived information, including osmotic ones. In the present study, we utilized immunohistochemistry to examine changes in the expression of NG2 and platelet-derived growth factor receptor beta (PDGFRB) in the OVLT, SFO and AP of adult mice during chronic osmotic stimulation. The expression of NG2 and PDGFRB was remarkably prominent in pericytes, although these angiogenesis-associated proteins are highly expressed at pericytes of developing immature vasculature. The chronic salt loading prominently increased the expression of NG2 in the OVLT and SFO and that of PDGFRB in the OVLT, SFO and AP. The vascular permeability of low-molecular-mass tracer fluorescein isothiocyanate was increased significantly by chronic salt loading in the OVLT and SFO but not AP. In conclusion, the present study demonstrates changes in pericyte expression of NG2 and PDGFRB and vascular permeability in the sensory CVOs by chronic osmotic stimulation, indicating active participation of the vascular system in osmotic homeostasis.

Screening assay for blood vessel maturation inhibitors.

In cancer patients, the development of resistance to anti-angiogenic agents targeting the VEGF pathway is common. Increased pericyte coverage of the tumor vasculature undergoing VEGF targeted therapy has been suggested to play an important role in resistance. Therefore, reducing the pericytes coverage of the tumor vasculature has been suggested to be a therapeutic approach in breaking the resistance to and increasing the efficacy of anti-angiogenic therapies. To screen compound libraries, a simple in vitro assay of blood vessel maturation demonstrating endothelial cells and pericytes association while forming lumenized vascular structures is needed. Unfortunately, previously described 3-dimensional, matrix based assays are laborious and challenging from an image and data acquisition perspective. For these reasons they generally lack the scalability needed to perform in a high-throughput environment. With this work, we have developed a novel in vitro blood vessel maturation assay, in which lumenized, vascular structures form in one optical plane and mesenchymal progenitor cells (10T1/2) differentiate into pericyte-like cells, which associate with the endothelial vessels (HUVECs). The differentiation of the 10T1/2 cells into pericyte-like cells is visualized using a GFP reporter controlled by the alpha smooth muscle actin promoter (SMP-8). The organization of these vascular structures and their recruited mural cells in one optical plane allows for automated data capture and subsequent image analysis. The ability of this assay to screen for inhibitors of pericytes recruitment was validated. In summary, this novel assay of in vitro blood vessel maturation provides a valuable tool to screen for new agents with therapeutic potential.

KIOM-79 protects AGE-induced retinal pericyte apoptosis via inhibition of NF-kappaB activation in vitro and in vivo.

KIOM-79 is an herbal mixture of parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhizae radix and Euphorbiae radix. In the present study, we determined the efficacy and possible mechanism of KIOM-79 on the advanced glycation end product (AGE)-modified bovine serum albumin (BSA)-induced apoptosis of cultured bovine retinal pericytes and rat retinal pericytes in Zucker diabetic fatty (ZDF) rats. Seven-week-old male ZDF rats were treated with KIOM-79 (50 mg/kg body weight) once a day orally for 13 weeks. KIOM-79 significantly inhibited pericyte apoptosis which were induced by the AGE-BSA treatment. The KIOM-79 treatment markedly suppressed the activation of nuclear factor-kappaB (NF-κB) through the inhibition of inhibitory κB kinase complex. In addition, the oral administration of KIOM-79 inhibited the changes in retinal vasculature (vascular hyperpermeability, acellular capillary). KIOM-79 strongly inhibited pericyte apoptosis, NF-κB activation and the expression of pro-apoptotic Bax and tumor necrosis factor-α. Our results suggest that KIOM-79 may exert inhibitory effects on AGE-induced pericyte apoptosis by blocking NF-κB activation, thereby ameliorating retinal microvascular dysfunction.

An intact kidney slice model to investigate vasa recta properties and function in situ.

BACKGROUND: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. METHODS: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in 'live' kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. RESULTS: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10-30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E(2)) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). CONCLUSIONS: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.

Folinic acid attenuates methotrexate chemotherapy-induced damages on bone growth mechanisms and pools of bone marrow stromal cells.

Chemotherapy often induces bone growth defects in pediatric cancer patients; yet the underlying cellular mechanisms remain unclear and currently no preventative treatments are available. Using an acute chemotherapy model in young rats with the commonly used antimetabolite methotrexate (MTX), this study investigated damaging effects of five once-daily MTX injections and potential protective effects of supplementary treatment with antidote folinic acid (FA) on cellular activities in the tibial growth plate, metaphysis, and bone marrow. MTX suppressed proliferation and induced apoptosis of chondrocytes, and reduced collagen-II expression and growth plate thickness. It reduced production of primary spongiosa bone, volume of secondary spongiosa bone, and proliferation of metaphyseal osteoblasts, preosteoblasts and bone marrow stromal cells, with the cellular activities being most severely damaged on day 9 and returning to or towards near normal levels by day 14. On the other hand, proliferation of marrow pericytes was increased early after MTX treatment and during repair. FA supplementation significantly suppressed chondrocyte apoptosis, preserved chondrocyte proliferation and expression of collagen-II, and attenuated damaging effects on production of calcified cartilage and primary bone. The supplementation also significantly reduced MTX effects on proliferation of metaphyseal osteoblastic cells and of bone marrow stromal cells, and enhanced pericyte proliferation. These observations suggest that FA supplementation effectively attenuates MTX damage on cellular activities in producing calcified cartilage and primary trabecular bone and on pools of osteoblastic cells and marrow stromal cells, and that it enhances proliferation of mesenchymal progenitor cells during bone/bone marrow recovery.

The role of polyol pathway in glucose-induced apoptosis of cultured retinal pericytes.

The pathogenesis of pericyte loss, an initial deficit in the early stage of diabetic retinopathy, remains unclear. Recent studies have suggested that polyol pathway hyperactivity and apoptosis may be involved in pericyte loss. The mechanisms of the glucose-induced apoptosis in retinal pericytes were investigated to evaluate the pathogenesis of diabetic retinopathy. Under the 20 mM glucose condition, intracellular calcium concentrations and caspase-3 activities were significantly increased, and reduced glutathione (GSH) contents were significantly decreased compared with those under the 5.5 mM glucose condition. These abnormalities were all significantly prevented by an aldose reductase inhibitor, SNK-860. Glucose-induced apoptosis was partially but significantly prevented by SNK-860, an inhibitor of calcium-dependent cysteine protease, calpain, or GSH supplementation, and completely normalized by a caspase-3 inhibitor. These observations suggest that glucose-induced apoptosis in retinal pericytes, as one of the pathogenic factors of diabetic retinopathy, would be mediated through an aldose reductase-sensitive pathway including calcium-calpain cascade and increased oxidative stress, and that caspase-3 would be located furthest downstream of these apoptotic signals.

Characterization of alpha-smooth muscle actin positive cells in mineralized human dental pulp cultures.

In response to injury, pulp precursor cells can differentiate into odontoblast-like cells that produce reparative dentine. In culture, pulp cells form mineralizing nodules, but the characteristics of the cells involved in this process are still not fully known. Human pulp cells for culture were obtained from coronal pulp isolated from non-erupted molars, and were maintained in RPMI 1640 medium supplemented with fetal calf serum. Nodules were forming in all human pulp primary cultures (HPPc) and human pulp subcultures observed until their fifth passage (HPSc<5). Mineralization of the nodules was confirmed by the presence of calcium and phosphate that were quantified by X-ray microanalysis. Specific immunolabeling revealed alpha-smooth muscle actin and vimentin in both HPPc and HPSc<5 cells. Cells positive for alpha-smooth muscle actin were either isolated or gathered together in the nodules. Under transmission electron microscopy, some cells in primary pulp cultures exhibited features typical of myofibroblasts or pericytes, such as stress fibers, fibronexus, indented nuclei and gap-junctions. These cells were frequently in close contact with mineral deposits. This work demonstrates for the first time the presence of pericytes or myofibroblasts in mineralized human pulp cultures, but further investigation is required to determine their origin, role and degree of differentiation.

Immune surveillance of mouse brain perivascular spaces by blood-borne macrophages.

Virchow-Robin's perivascular spaces lie between the basement membrane around pericytes and the basement membrane at the surface of the glia limitans of the brain vessels. They are directly connected to the subpial space and harbour a population of cells distinct from pericytes, perivascular microglia and other cells within perivascular spaces (e.g. T cells and mast cells) in their ability to quickly phagocytose particles from the cerebrospinal fluid (CSF). Morphology, function, and cell surface proteins of these perivascular cells suggest an origin from the monocyte/macrophage lineage. It is currently unclear to what extent these brain perivascular cells represent a resident population of histiocytes or undergo continuous supplementation from blood monocytes. Using transplants of green-fluorescent-protein (GFP)-transfected bone marrow cells, we therefore investigated the replacement of perivascular cells by blood-borne macrophages in adult mice. GFP-positive cells in the perivascular spaces were found as early as 2 weeks post transplantation. The substitution of host perivascular cells by donor-derived macrophages was then evaluated using immunocytochemistry and intraventricular injection of hydrophilic rhodamine-fluorescent tracers. Such tracers diffuse along perivascular spaces and are subsequently phagocytosed by perivascular cells leading to stable phagocytosis-dependent labelling. Thus, the population of newly immigrated macrophages could be related to the total number of perivascular macrophages. This approach revealed a continuous increase of donor-derived perivascular cells. At 14 weeks post transplantation, all perivascular cells were donor-derived. These data show that brain perivascular cells are a population of migratory macrophages and not resident histiocytes.

The ribosomal protein QM is expressed differentially during vertebrate endochondral bone development.

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein "QM." Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.