Ultrastructure of pericytes in early stages of histamine-induced inflammation

Physiological and ultrastructural assessment of changes in the walls of venules in the rat cremaster muscle after administration of histamine indicates that pericytes have essential roles in the normal functioning of venules during inflammation. Fluorescein-labelled albumin was used to quantitate macromolecular leakage and to select suitable venules for ultrastructural analysis 4 and 7 minutes after addition of histamine. Pericytes were concentrated over endothelial cell junctions and gaps. At 4 minutes, when albumin leakage was becoming detectable, gaps between endothelial cells were observed in the venule wall. In 24 serially sectioned gaps, pericytes formed covers, with contact points to the endothelial cells along the sides of the gaps. At 7 minutes, when albumin leakage was maximal, gaps with pericyte covers were still evident, but more commonly observed were pericyte covers over closed endothelial cell junctions. Spaces between the innermost pericytes and endothelial cells were enlarged by an order of magnitude, from 95 nm in controls to 872 nm at 4 minutes and 958 nm at 7 minutes. Pericytes formed coverings or bridges over inclusions of extravasated cells, fluid, proteins, and the vascular label monastral blue. The data indicate that pericytes protect the endothelial lining of venules during histamine-induced inflammation by forming a cohesive covering across gaps.     

Ultrastructure of pericytes in mouse heart

The pericytes of mouse myocardium are extensively branched cells that form an incomplete layer around the endothelium of capillaries and postcapillary venules. The membranes of pericytes and endothelial cells are connected by specialized junctions. Microtubules, intermediate (10-nm) filaments and microfilaments are oriented within circumferentially-arranged cytoplasmic processes of pericytes so as partially to encircle the endothelial cylinder. The intracellular organization of these myocardial pericytes suggests that they are smooth muscle-like cells which may be capable of influencing microvascular dynamics in the heart.     

Endothelial-pericyte interactions in angiogenesis

It takes two to make blood vessels—endothelial cells and pericytes. While the endothelial cells are the better characterized of the two, pericytes are now coming into focus as important regulators of angiogenesis and blood vessel function, and as potential drug targets. However, pericytes are still surrounded by much controversy. They are difficult to define, they constitute a heterogeneous population of cells, and their ontogeny is not well understood. They are plastic and have the capacity to differentiate into other mesenchymal cell types, such as smooth muscle cells, fibroblasts and osteoblasts. Recent interest in pericytes also stems from their potential involvement in diseases such as diabetic microangiopathy, tissue fibrosis, cancer, atherosclerosis and Alzheimer's disease. The present review focuses on the role of pericytes in physiological angiogenesis. The currently favored view states that the initial endothelial tubes form without pericyte contact, and that subsequent acquisition of pericyte coverage leads to vessel remodeling, maturation and stabilization. Improved means of identifying and visualizing pericytes now challenge this view and show that high numbers of pericytes invest in actively sprouting and remodeling vessels. Genetic data demonstrate the critical importance of pericytes for vascular morphogenesis and function, and imply specific roles for the cell type in various aspects of angiogenesis.The images were captured using a Leica confocal microscope, the purchase of which was made possible though a generous grant from the IngaBritt and Arne Lundberg's Research Foundation     

Descending vasa recta endothelium is an electrical syncytium

We examined gap junction coupling of descending vasa recta (DVR). DVR endothelial cells or pericytes were depolarized to record the associated capacitance transients. Virtually all endothelia and some pericytes exhibited prolonged transients lasting 10-30 ms. Carbenoxolone (100 microM) and 18beta-glycyrrhetinic acid (18betaGRA; 100 microM) markedly shortened the endothelial transients. Carbenoxolone and heptanol (2 mM) reduced the pericyte capacitance transients when they were prolonged. Lucifer yellow (LY; 2 mM) was dialyzed into the cytoplasm of endothelial cells and pericytes. LY spread diffusely along the endothelial monolayer, whereas in most pericytes, it was confined to a single cell. In some pericytes, complex patterns of LY spreading were observed. DVR cells were depolarized by voltage clamp as fluorescence of bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)] was monitored approximately 200 microm away. A 40-mV endothelial depolarization was accompanied by a 26.1 +/- 5.5-mV change in DiBAC(4)(3) fluorescence. DiBAC(4)(3) fluorescence did not change after 18betaGRA or when pericytes were depolarized. Similarly, propagated cytoplasmic Ca(2+) responses arising from mechanical perturbation of the DVR wall were attenuated by 18betaGRA or heptanol. Connexin (Cx) immunostaining showed predominant linear Cx40 and Cx43 in endothelia, whereas Cx37 stained smooth muscle actin-positive pericytes. We conclude that the DVR endothelium is an electrical syncytium and that gap junction coupling in DVR pericytes exists but is less pronounced.     

Peripheral nerve pericytes modify the blood–nerve barrier function and tight junctional molecules through the secretion of various soluble factors

The objectives of this study were to establish pure blood–nerve barrier (BNB) and blood–brain barrier (BBB)‐derived pericyte cell lines of human origin and to investigate their unique properties as barrier‐forming cells. Brain and peripheral nerve pericyte cell lines were established via transfection with retrovirus vectors incorporating human temperature‐sensitive SV40 T antigen (tsA58) and telomerase. These cell lines expressed several pericyte markers such as α‐smooth muscle actin, NG2, platelet‐derived growth factor receptor β, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, the inulin clearance was significantly lowered in peripheral nerve microvascular endothelial cells (PnMECs) through the up‐regulation of claudin‐5 by soluble factors released from brain or peripheral nerve pericytes. In particular, bFGF secreted from peripheral nerve pericytes strengthened the barrier function of the BNB by increasing the expression of claudin‐5. Peripheral nerve pericytes may regulate the barrier function of the BNB, because the BNB does not contain cells equivalent to astrocytes which regulate the BBB function. Furthermore, these cell lines expressed several neurotrophic factors such as NGF, BDNF, and GDNF. The secretion of these growth factors from peripheral nerve pericytes might facilitate axonal regeneration in peripheral neuropathy. Investigation of the characteristics of peripheral nerve pericytes may provide novel strategies for modifying BNB functions and promoting peripheral nerve regeneration. J. Cell. Physiol. 226: 255–266, 2010. © 2010 Wiley‐Liss, Inc.     

Perivascular cells for regenerative medicine

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.     

Cell contribution of vasa-vasorum to early arterial intimal thickening formation

In occluded femoral artery segments, intimal thickening occurred and abundant neovascularization from the surrounding microcirculation developed. Under these conditions, the contribution of vasa-vasorum as a source of supplementary population of cells during the early intimal thickening formation was studied. Using a technique that specifically labels venules, predominantly postcapillary venules, a marker-Monastral Blue B-was used as a tracer to follow the pericyte, endothelial cell and monocyte/macrophage lineages. In the first two days of the experiment, the marker was restricted to the wall of the periarterial microcirculation, being incorporated by endothelial cells, pericytes and some monocytes/macrophages crossing the venule walls. Later, the marker continues to be observed in some of the following cells: endothelial cells and pericytes of the newly-formed vessels, fibroblast-like cells, transitional cells between pericytes and fibroblast-like cells, macrophages migrating into the interstitium, myointimal cells and neoendothelial cells of the arterial lumen. These findings provide evidence that, during arterial intimal thickening formation in occluded arterial segments, the periarterial microvascularization contributes, in addition to recruited macrophages, newly-formed endothelial cells and a supplementary population of fibroblast-like cells and myointimal cells.     

Pericytes and vascular stability

Newly formed endothelial tubes are initially unstable and subsequently become stabilized through the formation of a perivascular matrix and the association with pericytes. The presence of pericyte per se is not sufficient for vascular stability. Instead, specific qualities of the cells are required that seem to correlate with marker expression and the nature of the endothelial-pericyte contacts. Most likely, specific intercellular signals are required as mediators of endothelial and pericyte cell function and vascular stability. Several ligand-receptor systems have been implicated in endothelial-pericyte interactions. Here, we discuss the role of some of these signaling systems in the regulation of vascular stability.     

The role of pericytes in angiogenesis

Pericytes are branched cells embedded within the basement membrane of capillaries and post-capillary venules. They provide an incomplete investment to endothelial cells, thus reinforcing vascular structure and regulating microvascular blood flow. Pericytes exert an important role on endothelial cell proliferation, migration and stabilization. Endothelial cells, in turn, stimulate expansion and activation of the pericyte precursor cell population. The balance between the number of endothelial cells and pericytes is highly controlled by a series of signaling pathway mechanisms operating in an autocrine and/or paracrine manner. In this review, we will first examine the molecular aspects of the pericyte activating factors secreted by endothelial cells, such as platelet derived growth factor B (PDGF-B), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β) and angiopoietins (Angs), as well as signaling pathways involving Notch and ephrins. We will then consider the complex and multivarious contribution of pericytes to the different aspects of angiogenesis with particular emphasis on the potential role of these cells as targets in tumor therapy.     

Immunocytochemical studies of desmin and vimentin in pericapillary cells of chicken

The composition of intermediate filaments in pericytes was examined by immunofluorescent and immunoelectron microscopic labeling of frozen sections of various chicken microvascular beds in situ. Pericytes in capillaries of cardiac muscle, exocrine pancreas, and kidney (peritubular capillary) were found to contain both desmin and vimentin. In some capillaries where pericytes do not exist, cells apposed to endothelial cells--the Ito cell in the hepatic sinusoid and the reticular cell in the splenic sinusoid--were shown to contain both of the intermediate filament proteins. In contrast, podocytes and mesangial cells around renal glomerular capillaries contained only vimentin. The presence of desmin supports the hypothesis that pericytes may have a contractile apparatus similar to that of vascular smooth muscle cells. Our results also revealed that even in microvascular beds where pericytes are not found, cells having both desmin and vimentin exist next to endothelial cells and may assume similar functions to pericytes.     

Microvascular pericytes contain muscle and nonmuscle actins

We have affinity-fractionated rabbit antiactin immunoglobulins (IgG) into classes that bind preferentially to either muscle or nonmuscle actins. The pools of muscle- and nonmuscle-specific actin antibodies were used in conjunction with fluorescence microscopy to characterize the actin in vascular pericytes, endothelial cells (EC), and smooth muscle cells (SMC) in vitro and in situ. Nonmuscle-specific antiactin IgG stained the stress fibers of cultured EC and pericytes but did not stain the stress fibers of cultured SMC, although the cortical cytoplasm associated with the plasma membrane of SMC did react with nonmuscle-specific antiactin. Whereas the muscle-specific antiactin IgG failed to stain EC stress fibers and only faintly stained their cortical cytoplasm, these antibodies reacted strongly with the fiber bundles of cultured SMC and pericytes. Similar results were obtained in situ. The muscle-specific antiactin reacted strongly with the vascular SMC of arteries and arterioles as well as with the perivascular cells (pericytes) associated with capillaries and post-capillary venules. The non-muscle-specific antiactin stained the endothelium and the pericytes but did not react with SMC. These findings indicate that pericytes in culture and in situ possess both muscle and nonmuscle isoactins and support the hypothesis that the pericyte may represent the capillary and venular correlate of the SMC.     

The impact of pericytes on the blood-brain barrier integrity depends critically on the pericyte differentiation stage

The blood-brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes and neurons. In this study we analyzed the differentiation stage dependent influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells. At first, we were able to induce two distinct differentiation stages of the primary pericytes in vitro. TGFβ treated pericytes expressed more α-SMA and actin while desmin, vimentin and nestin expression was decreased when compared to bFGF induced cells. Further analysis of α-SMA revealed that most of the pericytes differentiated with TGFβ expressed functional α-SMA while only few cells expressed functional α-SMA in the presence of bFGF. In addition the permeability factors VEGF, MMP-2 and MMP-9 were higher secreted by the α-SMA positive phenotype indicating a proangiogenic role of this TGFβ induced pericyte differentiation stage. Higher level of VEGF, MMP-2 and MMP-9 were as well detected in the TGFβ pretreated pericyte coculture with endothelial cells when compared to the influence of the bFGF pretreated pericytes. The TEER measurement of the barrier integrity of endothelial cells revealed that bFGF induced α-SMA negative pericytes stabilize the barrier integrity while α-SMA positive pericytes differentiated by TGFβ decrease the barrier integrity. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity in a differentiation stage dependant pathway.     

Pericytes in human breast stroma and the cells to which they relate.

The function of pericytes, the cells nearest the microvascular endothelium, has long been debated. On the basis of ultrastructural studies it is pointed out that they have specialised features in common with endothelial cells of the lymphatic labyrinth of the human breast. The latter are in continuity with the initial lymphatics. These features, which include points of stromal attachment, allow contraction or relaxation of the cell-processes. The cytoplasmic processes of the pericyte, when relaxed, form areas of contact with the blood vascular endothelium. Subsequent contraction may lead to loss of contact and increase in the area of vascular endothelium available for diffusion. The pericyte is thus equipped to act as a regulatory link between the blood vascular endothelium and that of the fine lymphatic system.     

Contractile elements in the regulation of macromolecular permeability

The leakage of macromolecules from the vasculature to the interstitium is greatly accentuated by mediators of edema such as histamine and bradykinin. The mechanism for this effect is not well delineated although many agents that affect smooth muscle tone may also affect macromolecular leakage. Leakage occurs primarily from the small venules. The demonstration that mediators of edema produce interendothelial gaps in the venules as well as changes in the shape of the endothelial nuclei has led to the hypothesis that a contraction of a vascular wall component may be responsible for the observed leakage of macromolecules. This component does not appear to be the vascular smooth muscle itself. Two other elements of the vascular wall, the endothelium and the pericytes, have been shown to contain many of the same elements of the contractile machinery present in smooth muscle. Most recent studies have presumed that endothelial cell contraction is responsible for the formation of the interendothelial gaps through which the macromolecules move. However, endothelial contraction has been difficult to demonstrate experimentally. Alternatively, inasmuch as pericytic processes can end near endothelial junctions and there is an abundance of fibronectin between the pericytes and the endothelium, it may be a pericytic contraction that causes the interendothelial gap formation.     

Ultrastructural localization of vesicle-associated membrane protein(s) to specialized membrane structures in human pericytes, vascular smooth muscle cells, endothelial cells, neutrophils, and eosinophils.

Vesicle-associated membrane proteins (VAMPs) are important to the trafficking of vesicles between membrane-bound intracytoplasmic organelles, in the facilitation of neurosecretion, and in constitutive and regulated secretion in non-neuronal cells. We used a pre-embedding ultrastructural immunonanogold method to localize VAMPs to subcellular sites in human cells of five lineages known to have cytoplasmic vesicles that may function in vesicular transport. We found VAMPs localized to caveolae in pericytes, vascular smooth muscle cells, and endothelial cells of venules, to the vesiculo-vacuolar organelle, recently defined in venular endothelial cells, to the vesicle-rich intergranular cytoplasm and secretory granule membranes of neutrophils, and to perigranular cytoplasmic secretory vesicles and secretory granule membranes in eosinophils. These specific localizations in five human vascular and granulocyte lineages support the notion that VAMPs have vesicle-associated functions in these cells.     

Peripheral nerve pericytes originating from the blood-nerve barrier expresses tight junctional molecules and transporters as barrier-forming cells

The objective of this study was to establish pure blood-nerve barrier (BNB)-derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier-forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines expressed several pericyte markers such as alpha-smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin-12, ZO-1, and ZO-2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO-1 were localized at the cell-cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans-pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell-cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood-brain barrier. Furthermore, we also showed that these cell lines expressed many barrier-related transporters. ABCG2, p-gp, MRP-1, and Glut-1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve-to-blood efflux and blood-to-peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis.     

Establishment of conditionally immortalized rat retinal pericyte cell lines (TR-rPCT) and their application in a co-culture system using retinal capillary endothelial cell line (TR-iBRB2)

The purpose of this study was to establish and characterize a retinal pericyte cell line from retinal capillaries of transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), and to apply this to the co-culture with a retinal capillary endothelial cell line. The conditionally immortalized rat retinal pericyte cell lines (TR-rPCTs), which express a temperature-sensitive large T-antigen, were obtained from two tsA58 Tg rats. These cell lines had a multicellular nodule morphology and reacted positively with von Kossa staining, a marker of calcification. TR-rPCTs cells expressed mRNA of pericyte markers such as rat intercellular adhesion molecule-1, platelet-derived growth factor-receptor beta, angiopoietin-1, and osteopontin. Western blot analysis indicated that alpha-smooth muscle actin (alpha-SMA) was expressed in TR-rPCT3 and 4 cells. In contrast, alpha-SMA was induced by transforming growth factor-beta1 and its enhancement was reduced by basic fibroblast growth factor in TR-rPCT1 and 2 cells. When TR-rPCT1 cells were cultured with a rat retinal endothelial cell line (TR-iBRB2) in a contact co-culture system, the number of TR-iBRB2 cells were significantly reduced in comparison with that of a single culture of TR-iBRB2 cells, suggesting that physical contact between pericytes and retinal endothelial cells is important for the growth of retinal endothelial cells. In conclusion, conditionally immortalized retinal pericyte cell lines were established from tsA58 Tg rats. These cell lines exhibited the properties of retinal pericytes and can be applied in co-culture systems with a retinal capillary endothelial cell line.     

Characterization of microvascular cell cultures from normotensive and hypertensive rat brains: pericyte-endothelial cell interactions in vitro

We used specific markers and fluorescence microscopy to identify and characterize cerebrovascular cells. Cultures were derived from brain microvessels isolated from normotensive (Wistar Kyoto, WKY) and spontaneously hypertensive (SHR) rat brains prior to, coincident with and following the onset of chronic hypertension. Endothelial cells were characterized using di-acyl LDL and non-muscle isoactin-specific antibodies. Cerebrovascular pericytes were identified with the anti-muscle and non-muscle actin antibody staining. Using this combination of cell culture and fluorescence localization, we have been able to demonstrate that brain pericytes are tightly associated with the endothelial cells of the hypertensive-prone and hypertensive cell cultures, but not with the normotensive endothelial cultures. While the endothelial-pericyte ratio in the hypertensive-prone microvascular cultures was between 5:1 and 10:1, the number of pericytes associated with the hypertensive rat brain cultures increased two to five times (2:1-1:1). Muscle and non-muscle actin antibody staining localized the spindle-shaped pericytes of the hypertensive microvascular colonies. Pericytes were found overlaying and encircling the endothelial cells. Normotensive pericytes were not endothelial-associated. Whereas the hypertensive pericyte is devoid of stress fibers, the normotensive pericyte is a larger, spread-out cell possessing numerous stress fibers rich in muscle and non-muscle actin. These results provide the first evidence that the etiology and inception of cerebrovascular disease may be pericyte-related and suggest that pericyte contraction could play a pivotal role in regulating the flow of blood within the brain microcirculation.     

Defective associations between blood vessels and brain parenchyma lead to cerebral hemorrhage in mice lacking alphav integrins

Mouse embryos genetically null for the alphav integrin subunit develop intracerebral hemorrhages at midgestation and die shortly after birth. A key question is whether the hemorrhage arises from primary defects in vascular endothelial cells or pericytes or from other causes. We have previously reported normal initiation of cerebral vessels comprising branched tubes of endothelial cells. Here we show that the onset of hemorrhage is not due to defects in pericyte recruitment. Additionally, most alphav-null vessels display ultrastructurally normal endothelium-pericyte associations and normal interendothelial cell junctions. Thus, endothelial cells and pericytes appear to establish their normal relationships in cerebral microvessels. However, by both light and electron microscopy, we detected defective associations between cerebral microvessels and the surrounding brain parenchyma, composed of neuroepithelial cells, glia, and neuronal precursors. These data suggest a novel role for alphav integrins in the association between cerebral microvessels and central nervous system parenchymal cells.