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.     

Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells

Morphological studies of developing capillaries and observations of alterations in capillaries associated with pathologic neovascularization indicate that pericytes may act as suppressors of endothelial cell (EC) growth. We have developed systems that enable us to investigate this possibility in vitro. Two models were used: a co-culture system that allowed direct contact between pericytes and ECs and a co-culture system that prevented physical contact but allowed diffusion of soluble factors. For these studies, co-cultures were established between bovine capillary ECs and the following growth-arrested cells (hereafter referred to as modulating cells): pericytes, smooth muscle cells (SMCs), fibroblasts, epithelial cells, and 3T3 cells. The modulating cell type was growth arrested by treatment with mitomycin C before co-culture with ECs. In experiments where cells were co-cultured directly, the effect of co-culture on EC growth was determined by comparing the mean number of cells in the co-cultures to the mean for each cell type (EC and modulating cell) cultured separately. Since pericytes and other modulating cells were growth arrested, any cell number change in co-cultures was due to EC growth. In the co-cultures, pericytes inhibited all EC proliferation throughout the 14-d time course; similar levels of EC inhibition were observed in SMC-EC co-cultures. Co-culture of ECs with fibroblasts, epithelial cells, and 3T3 cells significantly stimulated EC growth over the same time course (30-192% as compared to EC cultured alone). To determine if cell contact was required for inhibition, cells were co-cultured using Millicell chambers (Millipore Corp., Bedford, MA), which separated the cell types by 1-2 mm but allowed the exchange of diffusible materials. There was no inhibition of EC proliferation by pericytes or SMCs in this co-culture system. The influence of the cell ratios on observed inhibition was assessed by co-culturing the cells at EC/pericyte ratios of 1:1, 2:1, 5:1, 10:1, and 20:1. Comparable levels of EC inhibition were observed at ratios from 1:1 to 10:1. When the cells were co-cultured at a ratio of 20 ECs to 1 pericyte, inhibition of EC growth at 3 d was similar to that observed at other ratios. However, at higher ratios, the inhibition diminished so that by the end of the time course the co-cultured ECs were growing at the same rate as the controls. These results suggest that pericytes and SMCs can modulate EC growth by a mechanism that requires contact or proximity. We postulate that similar interactions may operate to modulate vascular growth in vivo.     

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.     

Microvascular remodeling and wound healing: A role for pericytes

Physiologic wound healing is highly dependent on the coordinated functions of vascular and non-vascular cells. Resolution of tissue injury involves coagulation, inflammation, formation of granulation tissue, remodeling and scarring. Angiogenesis, the growth of microvessels the size of capillaries, is crucial for these processes, delivering blood-borne cells, nutrients and oxygen to actively remodeling areas. Central to angiogenic induction and regulation is microvascular remodeling, which is dependent upon capillary endothelial cell and pericyte interactions. Despite our growing knowledge of pericyte-endothelial cell crosstalk, it is unclear how the interplay among pericytes, inflammatory cells, glia and connective tissue elements shape microvascular injury response. Here, we consider the relationships that pericytes form with the cellular effectors of healing in normal and diabetic environments, including repair following injury and vascular complications of diabetes, such as diabetic macular edema and proliferative diabetic retinopathy. In addition, pericytes and stem cells possessing "pericyte-like" characteristics are gaining considerable attention in experimental and clinical efforts aimed at promoting healing or eradicating ocular vascular proliferative disorders. As the origin, identification and characterization of microvascular pericyte progenitor populations remains somewhat ambiguous, the molecular markers, structural and functional characteristics of pericytes will be briefly reviewed.     

Ascorbic acid prevents high glucose-induced apoptosis in human brain pericytes

High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5 mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24 h with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 μM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant.     

Mechanisms controlling human endothelial lumen formation and tube assembly in three-dimensional extracellular matrices

Recent data have revealed new mechanisms that underlie endothelial cell (EC) lumen formation during vascular morphogenic events in development, wound repair, and other disease states. It is apparent that EC interactions with extracellular matrices (ECMs) establish signaling cascades downstream of integrin ligation leading to activation of the Rho GTPases, Cdc42 and Rac1, which are required for lumen formation. In large part, this process is driven by intracellular vacuole formation and coalescence, which rapidly leads to the creation of fluid-filled matrix-free spaces that are then interconnected via EC-EC interactions to create multicellular tube structures. EC vacuoles markedly accumulate in a polarized fashion directly adjacent to the centrosome in a region that strongly accumulates Cdc42 protein as indicated by green fluorescent protein (GFP)-Cdc42 during the lumen formation process. Downstream of Cdc42-mediated signaling, key molecules that have been identified to be required for EC lumen formation include Pak2, Pak4, Par3, Par6, and the protein kinase C (PKC) isoforms zeta and epsilon. Together, these molecules coordinately regulate the critical EC lumen formation process in three-dimensional (3D) collagen matrices. These events also require cell surface proteolysis mediated through membrane type 1 matrix metalloproteinase (MT1-MMP), which is necessary to create vascular guidance tunnels within the 3D matrix environment. These tunnels represent physical spaces within the ECM that are necessary to regulate vascular morphogenic events, including the establishment of interconnected vascular tube networks as well as the recruitment of pericytes to initiate vascular tube maturation (via basement membrane matrix assembly) and stabilization. Current research continues to analyze how specific molecules integrate signaling information in concert to catalyze EC lumen formation, pericyte recruitment, and stabilization processes to control vascular morphogenesis in 3D extracellular matrices.     

Hematopoietic Stem Cell Cytokines and Fibroblast Growth factor-2 Stimulate Human Endothelial Cell-Pericyte Tube Co-Assembly in 3D Fibrin Matrices under Serum-Free Defined Conditions

We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC) tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF)-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay) to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices) that regulates angiogenic events in postnatal life.     

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.     

Pericytes of the brain microvasculature express gamma-glutamyl transpeptidase.

The expression of gamma-glutamyl transpeptidase (GGT) is a specific property of the brain capillary endothelium that constitutes the blood-brain barrier. We report here the detection of GGT, not only in endothelial cells, but also in pericytes, demonstrating that a brain capillary-specific pericyte population exists. We raised antibodies to GGT using a porcine brain microvessel GGT-protein-A (staphylococcal protein A) fusion protein as antigen which was expressed in Escherichia coli. The immunohistochemical analysis of the subcapillary distribution of GGT in porcine brain cortex and cerebellum sections by both light and electron microscopy revealed the expression of GGT in the capillary-adjacent pericytes in addition to the GGT-positive endothelial layer. We confirmed these data for cultured porcine brain microvascular endothelial cells and pericytes. GGT immunofluorescence could be detected in both cell types in culture. Endothelial cells exhibited a weak staining, whereas pericytes were strongly positive for GGT. Due to the high phagocytotic activity of pericytes and their location on the abluminal surface of the microvessels, we propose a possible protective or detoxifying function of GGT in cerebrovascular pericytes.     

Glioblastoma: A Pathogenic Crosstalk between Tumor Cells and Pericytes

Cancers likely originate in progenitor zones containing stem cells and perivascular stromal cells. Much evidence suggests stromal cells play a central role in tumor initiation and progression. Brain perivascular cells (pericytes) are contractile and function normally to regulate vessel tone and morphology, have stem cell properties, are interconvertible with macrophages and are involved in new vessel formation during angiogenesis. Nevertheless, how pericytes contribute to brain tumor infiltration is not known. In this study we have investigated the underlying mechanism by which the most lethal brain cancer, Glioblastoma Multiforme (GBM) interacts with pre-existing blood vessels (co-option) to promote tumor initiation and progression. Here, using mouse xenografts and laminin-coated silicone substrates, we show that GBM malignancy proceeds via specific and previously unknown interactions of tumor cells with brain pericytes. Two-photon and confocal live imaging revealed that GBM cells employ novel, Cdc42-dependent and actin-based cytoplasmic extensions, that we call flectopodia, to modify the normal contractile activity of pericytes. This results in the co-option of modified pre-existing blood vessels that support the expansion of the tumor margin. Furthermore, our data provide evidence for GBM cell/pericyte fusion-hybrids, some of which are located on abnormally constricted vessels ahead of the tumor and linked to tumor-promoting hypoxia. Remarkably, inhibiting Cdc42 function impairs vessel co-option and converts pericytes to a phagocytic/macrophage-like phenotype, thus favoring an innate immune response against the tumor. Our work, therefore, identifies for the first time a key GBM contact-dependent interaction that switches pericyte function from tumor-suppressor to tumor-promoter, indicating that GBM may harbor the seeds of its own destruction. These data support the development of therapeutic strategies directed against co-option (preventing incorporation and modification of pre-existing blood vessels), possibly in combination with anti-angiogenesis (blocking new vessel formation), which could lead to improved vascular targeting not only in Glioblastoma but also for other cancers.     

Pericytes Regulate Vascular Basement Membrane Remodeling and Govern Neutrophil Extravasation during Inflammation

During inflammation polymorphonuclear neutrophils (PMNs) traverse venular walls, composed of the endothelium, pericyte sheath and vascular basement membrane. Compared to PMN transendothelial migration, little is known about how PMNs penetrate the latter barriers. Using mouse models and intravital microscopy, we show that migrating PMNs expand and use the low expression regions (LERs) of matrix proteins in the vascular basement membrane (BM) for their transmigration. Importantly, we demonstrate that this remodeling of LERs is accompanied by the opening of gaps between pericytes, a response that depends on PMN engagement with pericytes. Exploring how PMNs modulate pericyte behavior, we discovered that direct PMN-pericyte contacts induce relaxation rather than contraction of pericyte cytoskeletons, an unexpected response that is mediated by inhibition of the RhoA/ROCK signaling pathway in pericytes. Taking our in vitro results back into mouse models, we present evidence that pericyte relaxation contributes to the opening of the gaps between pericytes and to the enlargement of the LERs in the vascular BM, facilitating PMN extravasation. Our study demonstrates that pericytes can regulate PMN extravasation by controlling the size of pericyte gaps and thickness of LERs in venular walls. This raises the possibility that pericytes may be targeted in therapies aimed at regulating inflammation.     

Rat choroidal pericytes as a target of the autonomic nervous system

Pericytes are contractile cells that surround blood vessels. When contracting, they change the diameter of the vessel and therefore influence blood flow homeostasis; however, mechanisms controlling pericyte action are less well understood. Since blood flow regulation per se is controlled by the autonomic nervous system, the latter might also be involved in pericyte action. Hence, rat choroidal pericytes were analyzed for such a connection by using appropriate markers. Rat choroidal wholemounts and sections were prepared for immunohistochemistry of the pericyte marker chondroitin-sulfate-proteoglycan (NG2) and the pan-neuronal marker PGP9.5 or of tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP) and choline acetyl transferase (ChAT). Additionally, PGP9.5 and TH were analyzed in the choroid of DCX-dsRed2 transgenic rats, displaying red-fluorescent perivascular cells and serving as a putative model for studying pericyte function in vivo. Confocal laser-scanning microscopy revealed NG2-immunoreactive cells and processes surrounding the blood vessels. These NG2-positive cells were not co-localized with PGP9.5 but received close appositions of PGP9.5-, TH-, VIP- and ChAT-immunoreactive boutons and fibers. In the DCX-dsRed2 transgenic rat, PGP9.5 and TH were also densely apposed on the dsRed-positive cells adjacent to blood vessels. These cells were likewise immunoreactive for NG2, suggesting their pericyte identity. In addition to the innervation of vascular smooth muscle cells, the close relationship of PGP9.5 and further sympathetic (TH) and parasympathetic (VIP, ChAT) nerve fibers on NG2-positive pericytes indicated an additional target of the autonomic nervous system for choroidal blood flow regulation. Similar findings in the DCX-dsRed transgenic rat indicate the potential use of this animal model for in vivo experiments revealing the role of pericytes in blood flow regulation.     

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.     

Palmitate-induced apoptosis of microvascular endothelial cells and pericytes

BACKGROUND: Recent observations in the EURODIAB Complications Study demonstrated that markers of insulin resistance are strong risk factors for retinopathy incidence in patients with diabetes. However, the molecular mechanism underlying this remains to be elucidated. In this study, we investigated the influence of palmitate, a major saturated free fatty acid in plasma, on the apoptotic cell death of cultured microvascular endothelial cells (EC) and retinal pericytes. MATERIALS AND METHODS: The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H(2)DCFDA. DNA synthesis was determined by measuring [(3) H]-thymidine incorporation into cells. DNA fragmentations of EC were quantitatively analyzed in an enzyme-linked immunosorbent assay, and DNA laddering was evaluated on agarose gel electrophoresis. RESULTS: Palmitate increased ROS generation in microvascular EC. Furthermore, palmitate significantly inhibited DNA synthesis and induced apoptotic cell death in EC, which were completely prevented by an antioxidant, N-acetylcysteine. Palmitate up-regulated pericyte mRNA levels of a receptor for advanced glycation end products (AGE), and thereby potentiated the apoptotic effects of AGE on pericytes. CONCLUSIONS: The results suggest that palmitate could induce apoptotic cell death in microvascular EC and pericytes through the overgeneration of intracellular ROS, and thus be involved in the development of diabetic retinopathy.     

Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival

Pericytes have been suggested to play a role in regulation of vessel stability; one mechanism for this stabilization may be via pericyte-derived vascular endothelial growth factor (VEGF). To test the hypothesis that differentiation of mesenchymal cells to pericytes/smooth muscle cells (SMC) is accompanied by VEGF expression, we used endothelial cell (EC) and mesenchymal cell cocultures to model cell-cell interactions that occur during vessel development. Coculture of EC and 10T1/2 cells, multipotent mesenchymal cells, led to induction of VEGF expression by 10T1/2 cells. Increased VEGF expression was dependent on contact between EC-10T1/2 and was mediated by transforming growth factorbeta (TGFbeta). A majority of VEGF produced in coculture was cell- and/or matrix-associated. Treatment of cells with high salt, protamine, heparin, or suramin released significant VEGF, suggesting that heparan sulfate proteoglycan might be sequestering some of the VEGF. Inhibition of VEGF in cocultures led to a 75% increase in EC apoptosis, indicating that EC survival in cocultures is dependent on 10T1/2-derived VEGF. VEGF gene expression in developing retinal vasculature was observed in pericytes contacting newly formed microvessels. Our observations indicate that differentiated pericytes produce VEGF that may act in a juxtacrine/paracrine manner as a survival and/or stabilizing factor for EC in microvessels.     

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     

Optimization of use of UEA-1 magnetic beads for endothelial cell isolation

Most endothelial cells (EC) in the body belong to the microvasculature. Isolation and subsequent culture of these microvessel EC contributes greatly to our understanding of the heterogeneity and vascular specificity that exist between one organ site and another. However, a major obstacle is the overgrowth of contaminating cells (fibroblasts, pericytes, smooth-muscle cells) in cultures. Since 1990 the use of magnetic beads in combination with either a lectin, Ulex europaeus agglutinin-1 (UEA-1), or a monoclonal antibody has represented a powerful tool for the isolation/purification of microvessel EC. In the former case, operative conditions remain to be optimized to obtain pure cultures of EC.We have performed studies to optimize conditions of use for magnetic beads coated with UEA-1. Incubating beads with cells, the influences are studied of time, temperature, cell concentration, and number of beads per target cell for two cell types, human umbilical vein EC (HUVEC) and skin fibroblasts (HSF), either isolated or mixed. The effect of the last parameter was also checked on the behavior of cells undergoing proliferation after isolation. Results, expressed as isolation efficiency (from 40% to 90%) allowed us to select a 15-min incubation time at 4°C with rotary agitation, an optimal concentration of 4 x 10⁵ cells/ml, and an optimal cell:bead ration of 1:3. From a mixed cell population and in these conditions, even very low HUVEC:HSF proportions of 2.5:97.5 allowed us to obtain a pure HUVEC population in subsequent culture.Abbreviations UEA-1 Ulex europaeus agglutinin-1 - EC endothelial cells - HUVEC human umbilical vein endothelial cells - HSF human skin fibroblasts - MPC magnetic particle concentrator - IE isolation efficiency     

Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3

The endothelial cell (EC)-derived tissue inhibitor of metalloproteinase-2 (TIMP-2) and pericyte-derived TIMP-3 are shown to coregulate human capillary tube stabilization following EC-pericyte interactions through a combined ability to block EC tube morphogenesis and regression in three-dimensional collagen matrices. EC-pericyte interactions strongly induce TIMP-3 expression by pericytes, whereas ECs produce TIMP-2 in EC-pericyte cocultures. Using small interfering RNA technology, the suppression of EC TIMP-2 and pericyte TIMP-3 expression leads to capillary tube regression in these cocultures in a matrix metalloproteinase-1 (MMP-1)-, MMP-10-, and ADAM-15 (a disintegrin and metalloproteinase-15)-dependent manner. Furthermore, we show that EC tube morphogenesis (lumen formation and invasion) is primarily controlled by the TIMP-2 and -3 target membrane type (MT) 1 MMP. Additional targets of these inhibitors include MT2-MMP and ADAM-15, which also regulate EC invasion. Mutagenesis experiments reveal that TIMP-3 requires its proteinase inhibitory function to induce tube stabilization. Overall, these data reveal a novel role for both TIMP-2 and -3 in the pericyte-induced stabilization of newly formed vascular networks that are predisposed to undergo regression and reveal specific molecular targets of the inhibitors regulating these events.     

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.     

Generation and characterization of rgs5 mutant mice

Regulators of G-protein signaling (RGS) are involved in a wide variety of functions, including olfaction, vision, and cell migration. RGS5 has a perivascular expression pattern and was recently identified as a marker for brain pericytes. This suggests a role for RGS5 in vascular development and pericyte biology. We have created a mouse line which lacks the rgs5 gene and replaced it with a green fluorescent protein (GFP) reporter (rgs5(GFP/GFP)). The mice are viable and fertile and display no obvious developmental defects, and the vasculature appears to develop normally with proper pericyte coverage. Also, no differences were observed in the vasculature under pathological conditions, such as tumor growth and oxygen-induced retinopathy. The GFP expression in pericytes of rgs5(GFP) mice allows detection and sorting of these cells, thereby providing a valuable novel tool for pericyte research.