Follicular dendritic cells emerge from ubiquitous perivascular precursors.

The differentiation of follicular dendritic cells (FDC) is essential to the remarkable microanatomic plasticity of lymphoid follicles. Here we show that FDC arise from ubiquitous perivascular precursors (preFDC) expressing platelet-derived growth factor receptor ß (PDGFRß). PDGFRß-Cre-driven reporter gene recombination resulted in FDC labeling, whereas conditional ablation of PDGFRß(+)-derived cells abolished FDC, indicating that FDC originate from PDGFRß(+) cells. Lymphotoxin-α-overexpressing prion protein (PrP)(+) kidneys developed PrP(+) FDC after transplantation into PrP(-) mice, confirming that preFDC exist outside lymphoid organs. Adipose tissue-derived PDGFRß(+) stromal-vascular cells responded to FDC maturation factors and, when transplanted into lymphotoxin ß receptor (LTßR)(-) kidney capsules, differentiated into Mfge8(+)CD21/35(+)FcγRIIß(+)PrP(+) FDC capable of trapping immune complexes and recruiting B cells. Spleens of lymphocyte-deficient mice contained perivascular PDGFRß(+) FDC precursors whose expansion required both lymphoid tissue inducer (LTi) cells and lymphotoxin. The ubiquity of preFDC and their strategic location at blood vessels may explain the de novo generation of organized lymphoid tissue at sites of lymphocytic inflammation.

Apolipoprotein E controls cerebrovascular integrity via cyclophilin A.

Human apolipoprotein E has three isoforms: APOE2, APOE3 and APOE4. APOE4 is a major genetic risk factor for Alzheimer's disease and is associated with Down's syndrome dementia and poor neurological outcome after traumatic brain injury and haemorrhage. Neurovascular dysfunction is present in normal APOE4 carriers and individuals with APOE4-associated disorders. In mice, lack of Apoe leads to blood-brain barrier (BBB) breakdown, whereas APOE4 increases BBB susceptibility to injury. How APOE genotype affects brain microcirculation remains elusive. Using different APOE transgenic mice, including mice with ablation and/or inhibition of cyclophilin A (CypA), here we show that expression of APOE4 and lack of murine Apoe, but not APOE2 and APOE3, leads to BBB breakdown by activating a proinflammatory CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes. This, in turn, leads to neuronal uptake of multiple blood-derived neurotoxic proteins, and microvascular and cerebral blood flow reductions. We show that the vascular defects in Apoe-deficient and APOE4-expressing mice precede neuronal dysfunction and can initiate neurodegenerative changes. Astrocyte-secreted APOE3, but not APOE4, suppressed the CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes through a lipoprotein receptor. Our data suggest that CypA is a key target for treating APOE4-mediated neurovascular injury and the resulting neuronal dysfunction and degeneration.

Pericytes regulate the blood-brain barrier.

The blood-brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB.

Endothelial-mural cell signaling in vascular development and angiogenesis.

Mural cells are essential components of blood vessels and are necessary for normal development, homeostasis, and organ function. Alterations in mural cell density or the stable attachment of mural cells to the endothelium is associated with several human diseases such as diabetic retinopathy, venous malformation, and hereditary stroke. In addition mural cells are implicated in regulating tumor growth and have thus been suggested as potential antiangiogenic targets in tumor therapy. In recent years our knowledge of mural cell function and endothelial-mural cell signaling has increased dramatically, and we now begin to understand the mechanistic basis of the key signaling pathways involved. This is mainly thanks to sophisticated in vivo experiments using a broad repertoire of genetic technologies. In this review, we summarize the five currently best understood signaling pathways implicated in mural cell biology. We discuss PDGFB/PDGFRbeta- dependent pericyte recruitment, as well as the role of angiopoietins and Tie receptors in vascular maturation. In addition, we highlight the effects of sphingosine-1-phosphate signaling on adherens junction assembly and vascular stability, as well as the role of TGF-beta-signaling in mural cell differentiation. We further reflect recent data suggesting an important function for Notch3 signaling in mural cell maturation.

Identification of a core set of 58 gene transcripts with broad and specific expression in the microvasculature.

OBJECTIVE: Pathological angiogenesis is an integral component of many diseases. Antiangiogenesis and vascular targeting are therefore promising new therapeutic principles. However, few endothelial-specific putative drug targets have been identified, and information is still limited about endothelial-specific molecular processes. Here we aimed at determining the endothelial cell-specific core transcriptome in vivo. METHODS AND RESULTS: Analysis of publicly available microarray data identified a mixed vascular/lung cluster of 132 genes that correlated with known endothelial markers. Filtering against kidney glomerular/nonglomerular and brain vascular/nonvascular microarray profiles separated contaminating lung markers, leaving 58 genes with broad and specific microvascular expression. More than half of these have not previously been linked to endothelial functions or studied in detail before. The endothelial cell-specific expression of a selected subset of these, Eltd1, Gpr116, Ramp2, Slc9a3r2, Slc43a3, Rasip1, and NM_023516, was confirmed by real-time quantitative polymerase chain reaction and/or immunohistochemistry. CONCLUSIONS: We have used a combination of publicly available and own microarray data to identify 58 gene transcripts with broad yet specific expression in microvascular endothelium. Most of these have unknown functions, but many of them are predicted to be cell surface expressed or implicated in cell signaling processes and should therefore be explored as putative microvascular drug targets.

Endothelial/pericyte interactions.

Interactions between endothelial cells and mural cells (pericytes and vascular smooth muscle cells) in the blood vessel wall have recently come into focus as central processes in the regulation of vascular formation, stabilization, remodeling, and function. Failure of the interactions between the 2 cell types, as seen in numerous genetic mouse models, results in severe and often lethal cardiovascular defects. Abnormal interactions between the 2 cell types are also implicated in a number of human pathological conditions, including tumor angiogenesis, diabetic microangiopathy, ectopic tissue calcification, and stroke and dementia syndrome CADASIL. In the present review, we summarize current knowledge concerning the identity, characteristics, diversity, ontogeny, and plasticity of pericytes. We focus on the advancement in recent years of the understanding of intercellular communication between endothelial and mural cells with a focus on transforming growth factor beta, angiopoietins, platelet-derived growth factor, spingosine-1-phosphate, and Notch ligands and their respective receptors. We finally highlight recent important data contributing to the understanding of the role of pericytes in tumor angiogenesis, diabetic retinopathy, and hereditary lymphedema.

The integrin beta1 subunit transmembrane domain regulates phosphatidylinositol 3-kinase-dependent tyrosine phosphorylation of Crk-associated substrate.

Our previous studies on the transmembrane domain of human integrin subunits have shown that a conserved basic amino acid in both subunits of integrin heterodimers is positioned in the plasma membrane in the absence of interacting proteins. To investigate the possible functional role of the lipid-embedded lysine in the mouse integrin beta1 subunit, this amino acid was replaced with leucine, and the mutated beta1 subunit (beta1A(K756L)) was stably expressed in beta1-deficient GD25 cells. The extracellular domain of beta1A(K756L) integrins possesses a competent conformation for ligand binding as determined by the ability to mediate cell adhesion, and by the presence of the monoclonal antibody 9EG7 epitope. However, the spreading of GD25-beta1A(K756L) cells on fibronectin and laminin-1 was impaired, and the rate of migration of GD25-beta1A(K756L) cells on fibronectin was reduced compared with GD25-beta1A cells. Phosphorylation of tyrosines in focal adhesion kinase (FAK) and the Y416 in c-Src in response to beta1A(K756L)-mediated adhesion was similar to that induced by wild-type beta1. The tyrosine phosphorylation level of paxillin, a downstream target of FAK/Src, was unaffected by the beta1 mutation, whereas tyrosine phosphorylation of CAS was strongly reduced. The results demonstrate that CAS is a target for phosphorylation both by FAK-dependent and -independent pathways after integrin ligation. The latter pathway was inhibited by wortmannin and LY294002, implicating that it required an active phosphatidylinositol 3-kinase. Furthermore, the K756L mutation in the beta1 subunit was found to interfere with beta1-induced activation of Akt. The results from this study identify phosphatidylinositol 3-kinase as an early component of a FAK-independent integrin signaling pathway triggered by the membrane proximal part of the beta1 subunit.

Splice variants of human beta 1 integrins: origin, biosynthesis and functions.

The integrin beta1 subfamily of adhesion receptors consists of 12 members and forms the biggest subfamily among integrins. Human integrin subunit beta1 has five cytoplasmic splice variants (beta1A, beta1B, beta1C-1, beta1-C2, beta1D). Even though cytoplasmic splice variants do not change the ligand-specificity of a beta1 integrin, clustering of these different splice variants triggers signaling pathways that lead to a different cellular response. The main focus of this review is on the origin and specific functions of the less abundant human integrin beta1 splice variants (B, C-1, C-2, D).

Getting to know the cast - cellular interactions and signaling at the neurovascular unit.

The neurovascular unit (NVU), consisting of endothelial cells, basement membrane, pericytes, astrocytes and microglial cells, couples local neuronal function to local cerebral blood flow and regulates transport of blood-borne molecules across the blood-brain barrier (BBB). The building blocks and the phenotype of the NVU are well-established but the intercellular signaling between the different components remains elusive. A better understanding of the cellular interactions and signaling within the NVU is critical for the development of efficient therapeutics for the treatment of a variety of brain diseases, such as brain cancer, stroke, neuroinflammation and neurodegeneration. This review gives an overview about the current in vivo knowledge of the NVU and the communication between its different cellular constituents. We also discuss the usefulness of various model organisms for studies of the brain vasculature.

Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.

Pericytes, the mural cells of blood microvessels, have recently come into focus as regulators of vascular morphogenesis and function during development, cardiovascular homeostasis, and disease. Pericytes are implicated in the development of diabetic retinopathy and tissue fibrosis, and they are potential stromal targets for cancer therapy. Some pericytes are probably mesenchymal stem or progenitor cells, which give rise to adipocytes, cartilage, bone, and muscle. However, there is still confusion about the identity, ontogeny, and progeny of pericytes. Here, we review the history of these investigations, indicate emerging concepts, and point out problems and promise in the field of pericyte biology.

Pericytes and the blood-brain barrier: recent advances and implications for the delivery of CNS therapy.

"Once the regulation of brain endothelial transcytosis is understood at the molecular level, it should be possible to exploit these mechanisms as targets for facilitated CNS drug delivery".

PDGF-B signaling is important for murine cardiac development: its role in developing atrioventricular valves, coronaries, and cardiac innervation.

We hypothesized that PDGF-B/PDGFR-beta-signaling is important in the cardiac contribution of epicardium-derived cells and cardiac neural crest, cell lineages crucial for heart development. We analyzed hearts of different embryonic stages of both Pdgf-b-/- and Pdgfr-beta-/- mouse embryos for structural aberrations with an established causal relation to defective contribution of these cell lineages. Immunohistochemical staining for alphaSMA, periostin, ephrinB2, EphB4, VEGFR-2, Dll1, and NCAM was performed on wild-type and knockout embryos. We observed that knockout embryos showed perimembranous and muscular ventricular septal defects, maldevelopment of the atrioventricular cushions and valves, impaired coronary arteriogenesis, and hypoplasia of the myocardium and cardiac nerves. The abnormalities correspond with models in which epicardial development is impaired and with neuronal neural crest-related innervation deficits. This implies a role for PDGF-B/PDGFR-beta-signaling specifically in the contribution of these cell lineages to cardiac development.

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.

Platelet-derived growth factor receptor-beta promotes early endothelial cell differentiation.

Platelet-derived growth factor BB (PDGF-BB) has been assigned a critical role in vascular stability by promoting the recruitment of PDGF receptor-beta-expressing perivascular cells. Here we present data indicating that early hematopoietic/endothelial (hemangio) precursors express PDGFR-beta based on coexpression with CD31, vascular endothelial growth factor receptor-2, and CD41 in 2 models: mouse yolk sac (embryonic day 8 [E8]) and differentiating mouse embryonic stem cells (embryoid bodies). Expression of PDGFR-beta on hemangioprecursor cells in the embryoid bodies gradually disappeared, and, at E14, expression appeared on perivascular cells. Activation of the PDGFR-beta on the hemangioprecursors accelerated the differentiation of endothelial cells, whereas differentiation of the hematopoietic lineage was suppressed. In E9.5 yolk sacs derived from recombinant mice expressing kinase-active PDGFR-beta with an aspartic acid to asparagine (D894N) replacement in the kinase activating loop and from mice with ubiquitous expression of PDGF-BB driven by the Rosa26 locus, the number of CD41-expressing early hematopoietic cells decreased by 36% and 34%, respectively, compared with staged wild-type littermates. Moreover, enhanced vascular remodeling was evident in the Rosa26-PDGF-BB yolk sacs. We conclude that PDGFR-beta is expressed on early hemangioprecursor cells, regulating vascular/hematopoietic development.

Ephrin-A2 reverse signaling negatively regulates neural progenitor proliferation and neurogenesis.

The number of cells in an organ is regulated by mitogens and trophic factors that impinge on intrinsic determinants of proliferation and apoptosis. We here report the identification of an additional mechanism to control cell number in the brain: EphA7 induces ephrin-A2 reverse signaling, which negatively regulates neural progenitor cell proliferation. Cells in the neural stem cell niche in the adult brain proliferate more and have a shorter cell cycle in mice lacking ephrin-A2. The increased progenitor proliferation is accompanied by a higher number of cells in the olfactory bulb. Disrupting the interaction between ephrin-A2 and EphA7 in the adult brain of wild-type mice disinhibits proliferation and results in increased neurogenesis. The identification of ephrin-A2 and EphA7 as negative regulators of progenitor cell proliferation reveals a novel mechanism to control cell numbers in the brain.

Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits.

Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits alpha(2), alpha(5), beta(1), and beta(2) is embedded in the plasma membrane in the absence of interacting proteins (Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030-37034). Using a glycosylation mapping technique, we here show that alpha(10) and beta(8), two subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were found to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-terminal end of the transmembrane (TM) domain seems likely to be general for all 24 integrin subunits. Furthermore, we have determined the N-terminal border of the TM domains of the alpha(2), alpha(5), alpha(10), beta(1), and beta(8) subunits. The TM domain of subunit beta(8) is found to be 22 amino acids long, with a second basic residue (Arg(684)) positioned just inside the membrane at the exoplasmic side, whereas the lipidembedded domains of the other subunits are longer, varying from 25 (alpha(2)) to 29 amino acids (alpha(10)). These numbers implicate that the TM region of the analyzed integrins (except beta(8)) would be tilted or bent in the membrane. Integrin signaling by transmembrane conformational change may involve alteration of the position of the segment adjacent to the conserved lysine. To test the proposed "piston" model for signaling, we forced this region at the C-terminal end of the alpha(5) and beta(1) TM domains out of the membrane into the cytosol by replacing Lys-Leu with Lys-Lys. The mutation was found to not alter the position of the N-terminal end of the TM domain in the membrane, indicating that the TM domain is not moving as a piston. Instead the shift results in a shorter and therefore less tilted or bent TM alpha-helix.

Role of the beta1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia.

Invasin of Yersinia pseudotuberculosis binds to beta1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the beta1-integrin-mediated internalization of Yersinia, a beta1-integrin-deficient cell line, GD25, transfected with wild-type beta1A, beta1B or different mutants of the beta1A subunit was used. Both beta1A and beta1B bound to invasin-expressing bacteria, but only beta1A was able to mediate internalization of the bacteria. The cytoplasmic region of beta1A, differing from beta1B, contains two NPXY motifs surrounding a double threonine site. Exchanging the tyrosines of the two NPXYs to phenylalanines did not inhibit the uptake, whereas a marked reduction was seen when the first tyrosine (Y783) was exchanged to alanine. A similar reduction was seen when the two nearby threonines (TT788-9) were exchanged with alanines. It was also noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex structures was unable to mediate uptake of invasin-expressing bacteria.