Current Immunology Reviews, 2012, 8, 227-247 227 Vascular Endothelium and Vector Borne Pathogen Interactions Moez Berrich*,1, Henri-Jean Boulouis1, Martine Monteil1, Claudine Kieda2 and Nadia Haddad*,1

1UPE, Ecole Nationale Vétérinaire d'Alfort, UMR BIPAR, ENVA, ANSES, UPEC, USC INRA, 23, rue du Gl de Gaulle - 94703 Maisons-Alfort, France 2Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique, Unité Propre de Recherche 4301, 45045 Orléans, France

Abstract: The endothelium is the thin layer of cells that lines the lumen of blood and lymphatic vessels. Endothelial cells (ECs) from different locations have distinct and characteristic expression patterns that persist during in vitro culture. Although gene expression patterns in cultured cells clearly reveal the molecular heterogeneity of these ECs, their corelation with their in vivo counterparts remains to be defined. Situated at the interface between blood and tissues, the endothelium plays a central role for critical functions and represents a physical barrier for both blood-borne pathogens and immune cells, which must cross this barrier for trafficking between the bloodstream and tissues. Endothelial cells are target cells for several infectious agents, including Anaplasma, , Orientia and Rickettsia. The lack of appropriate spontaneous or experimentally-induced animal models is a serious limitation for the study of pathogen-ECs interactions and is the major justification for the use of ECs cultures. Bartonella adherence to ECs is mediated by type-IV like pili and some outer membrane proteins. Some differences exist among Bartonella species adherence mechanisms. ECs are invaded either by an endocytic uptake or by engulfment of Bartonella. The activation of ECs by chronic inflammation and direct or indirect action of some Bartonella species leads to proliferation, angiogenesis and vasoproliferative tumor growth. Interactions between Anaplasma, Orientia, Rickettsia and ECs are less documented than Bartonella but differ significantly from the mechanisms described for Bartonella. At the least, they do not induce vasoproliferation. This review summarizes our understanding of the diversity of ECs, the vector borne bacterium-ECs interactions and the mechanisms of bacterial virulence and persistence. Keywords: Anaplasma, angiogenesis, apoptosis, Bartonella, endothelial cells, heterogeneity, interactions, macrophages, organ specificity, Orientia, Rickettsia, tumor, vascular endothelium, vector borne , VEGF.

INTRODUCTION Accordingly, in vitro evidence of ECs infection is not sufficient, but can contribute to clarify some pathological The endothelium is a single layer of endothelial cells mechanisms. The lack of appropriate spontaneous or (ECs) that lines the lumen of blood and lymphatic vessels. experimentally-induced animal models is a serious limitation ECs are very heterogeneous and constitute diverse for the study of pathogen-ECs interactions and is the major populations with great differences [1] according to the type justification for the use of ECs cultures. of vessel and the organ they belong to and the biological state [2]. The endothelium is not a passive barrier and plays During infection within hosts the ECs-bacteria important roles in the development and remodeling of interactions result into a broad range of clinical vasculature, maintenance of vascular tone, blood fluidity, manifestations. The list of bacteria that have been found able coagulation, nutrient exchange, homeostasis, angiogenesis, to subvert certain cellular functions, resulting in cell organ development and inflammation [3]. Moreover, ECs invasion, proinflamatory activation and modulation of  represent a physical barrier to both blood-borne pathogens apoptosis is limited to some - (Fig. 1), the and immune cells which must cross this barrier for majority of which belong to the order of . This trafficking between tissues and the bloodstream. order is composed by two families, the (genus Rickettsia and Orientia) and the (that ECs are target cells for several infectious agents includes the genus Anaplasma, Ehrlichia, and including some . Valbuena and Walker Wolbachia) [5]. Anaplasma-infected ECs lead to unique [4] have proposed the three following criteria as necessary pathogen-specific host cell functional alterations that are for considering the relationships between pathogenic bacteria likely to be important for pathogen survival, pathogenesis and ECs as significant in the pathological process: Bacteria and human disease induction [6]. Orientia causes vasculitis intracellular location in the ECs of natural hosts; Bacteria in humans by replicating inside macrophages and ECs [7]. must be able to multiply in ECs and the endothelium must be Rickettsiae cause life threatening spotted fevers and a consistent target of the infection during the disease. fevers via bacterial transmission by arthropod inoculation into skin, followed by hematogenous spread and disseminated ECs infection [4]. Some members of another *Address correspondence to this author at the Ecole Nationale Vétérinaire d'Alfort - UMR BIPAR, ENVA, ANSES, UPEC, USC INRA, LERPAZ, 7 family, the Bartonellaceae, not belonging to the Rickettsiales avenue du Général de Gaulle, 94704 Maisons-Alfort, France; order, but closer to the genera Agrobacterium, Brucella, and Tel: 00 33 1 43 96 73 15; Fax: 00 33 1 43 96 71 31; Rhizobium can trigger massive proliferation of ECs, leading E-mails: [email protected]; [email protected] to vascular tumor formation [8-9].

1875-631X/12 $58.00+.00 © 2012 Bentham Science Publishers 228 Current Immunology Reviews, 2012, Vol. 8, No. 3 Berrich et al.

Fig. (1). Different degrees of consequences of intra-cellular infection by bacteria that are able to replicate in ECs. As shown in the figure, the bacteria that are able to induce an anti-apoptotic effect (2) are a fortiori able to induce a persistant bacteremia (1), and the bacteria that are able to induce an angiogenic effect (3) are also able to induce an anti-apoptotic effect. *In various blood cells according to the genus.

Understanding the mechanisms that contribute to highly complex topology of the endothelium. The pathogens/endothelium interactions is essential to approach understanding of this complexity improved thanks to the in the processes by which infectious agents penetrate the vitro models of endothelium. Immunohistochemical analyses endothelial barrier and to control the clinical outcome of showed that antigen expression and glycosylation patterns such infectious diseases. are different according to the different organs and tissues where the ECs are originating from [14-20]. This review is dedicated to the study of these interactions and will not consider the sepsis mediated endothelial injury. Bizouarne et al. [21] have demonstrated the fine difference between ECs from the high endothelial ORGAN SPECIFICITY AND HETEROGENEITY OF postcapillary venules (HEV) in peripheral lymph nodes ENDOTHELIAL CELLS (PLN) compared to the cells from the post capillary veinules of the Peyer’s Patches (PP) at the molecular and functional ECs are morphologically and functionally heterogeneous levels. These cells from the HEV in their respective with major differences between those from the macro- versus secondary lymphoid organs, are responsible for the selection micro-circulation as documented for a variety of tissues [10]. of distinct lymphocyte populations. As such, they are They differ morphologically, in size, shape, thickness, regulating the homing process of lymphocytes during the number of microvilli, and position of the nucleus [11] and immune response. Bizouarne et al. [22] and Denis et al. [23] functionally by differences in the released substances and in demonstrated that human ECs from PLN and PP are their interactions with leukocytes and pathogens [12]. responsible for distinct gene expression of adhesion molecules and distinct expression of addressins as well as This heterogeneous population of vascular endothelium distinct endogenous lectin expression and activity. The cells specializes in response to genetic programs and “organo specificity” of the endothelium was proven by environmental factors, which contribute to their ability to Kieda et al. [2] who have established endothelial in vitro play distinct roles in different vessels, tissues, organs, and models representative of the various lymphoid and response to stresses and infection [11]. The endothelium phenotypic diversity reflects the biological needs of the peripheral tissues (Table 1). Moreover, Lamerant and Kieda [24] have used the differential gene expression display to underlying tissue as illustrated by the five distinct ECs types identify endothelial genes that are distinctively expressed and phenotypes of the heart (endocardial, coronary arterial, between those ECs from distinct tissue origin. venous, capillary, and lymphatic). Coronary ECs establish a typical network throughout the myocardium, whereas Several studies support this concept. For example, the endocardial ECs form a large epithelial sheet with no plasma glycoprotein known as von Willebrand factor, is sprouting into the myocardium [13]. prominent in veins, less prominent in arteries, and largely absent from sinusoidal ECs; the endothelial protein C While the cultured ECs became a focal point for research receptor is predominantly expressed by large vessel ECs in vascular biology, increasing evidence was pointing to the [25]; and the cell-surface signaling proteins ephrin-B2 and Vascular Endothelium and Vector Borne Pathogen Interactions Current Immunology Reviews, 2012, Vol. 8, No. 3 229

Table 1. Organo-Specificity of Human Endothelial Cells as Demonstrated by the Presence of Different Cell Surface Markers [2]

Cells Characterisation of Human Endothelial Cells Markers

Markers VE-cadherin CD62E CD62P CD34 CD15s PNaD.JG1 MAdCAM-1 CD54 CD29 CD49E Cd44

HPLNEC.B3 peripheral + + - + + + - - + - - HPLNEC.S1R1 peripheral + + - + + + - - + - - HOMEC.J6B ovary + + - + + + - - + - - HLMEC lung + + ------+ - - HSKMEC.1 skin + + + - - - - + + + - HLMNEC.MEL illeal + + - - - - + + + - - HAPEC.S1 appendix + + + - - - + + + + HIMEC.1 intestine + + + - - - + + + + HPLNEC.B3 & HPLNEC.S1R1: Human Peripheral Lymph Node EC (from respectively Hodgkin’s and non Hodgkin’s lymphomas); HOMEC.J6B: Human Ovary Microvascular EC (from ovarian carcinoma)); HLMEC: Human Lung Microvascular Endothelial Cell (from normal lung); HSKMEC.1: Human Skin Microvascular EC (from normal skin); HMLNEC.MEL: Human Mesentiric Lymph Node EC (from a metastatic malignant melanoma); HAPEC.S1: Human Appendix EC (from a patient with appendicitis); HIMEC.1: Human Intestine Microvascular EC (from normal intestine). ephrin-B4 are specifically expressed by arteries and veins, The most prominent structure-function genes are related to respectively [26]. intercellular junctional contacts. Small capillaries with tight, continuous junctions are more present in the central nervous In addition, a high level of secretion of vasoactive system, providing brain protection towards bacterial or toxic substances (angiotensin-II and thromboxane A2) was insults. Thicker capillaries, also with continuous endothelium, observed in PLEC (microvascular ECs of the human full- are typically found in skeletal tissues and in the heart, testes and term placenta) and MIEC (human dermal microvessels ECs) ovaries. Discontinuous ECs, with gaps of variable size between as compared to HUVEC (Human Umbilical Vein Endothelial Cells), clearly demonstrating the physiological cells (fenestrations), allow efficient transit of macromolecules and thus are predominant in endocrine glands and in the kidney heterogeneity of ECs [1]. This correlates with the increased [11]. The factors that contribute to the morphology and function expression of angiotensin-converting enzyme activity, in of the intercellular contacts and that determine the formation of MIEC versus HUVEC [27]. tight junctions versus fenestrations are occludins, claudins, zona Furthermore, mitogenic activity of endothelial growth occludens proteins, and the family of junctional adhesion factors is also tissue-specific [28-30]. Microvascular ECs molecules [34-36]. were more susceptible to growth factors than macrovascular A recent study examined the mechanisms for segment- ECs [31]. Moreover, kinetics of cytokine activity is specific barrier functions between lung microvascular and depending of the type of ECs. In HUVEC, VEGF (Vascular artery ECs. Transendothelial electrical resistance was Endothelial Growth Factors) and FGF-2 (Fibroblast Growth significantly higher in microvascular barriers as compared to Factor-2) peak after 24 H and decrease by 48H whereas kinetics were reversed in PLEC and MIEC [1]. Nevertheless, the arterial barrier. The N-cadherin and the activated leukocyte cell adhesion molecule (ALCAM) were more most studies related to the mitogenic effects of endothelial expressed in microvascular than in pulmonary artery ECs. growth factors have been carried out on macrovasculature ALCAM was recruited to cell junctions in pulmonary endothelium, notably HUVEC. microvascular ECs but remained predominantly cytosolic in Chi et al. [3] have used microarray techniques on a pulmonary artery ECs [37]. ‘global’ scale and have evaluated the expression profile of 53 In addition, microvascular ECs express higher levels of cultured human endothelial cell lines from veins, arteries and proteins involved in the traffic of circulating blood cells and microvessels from 14 different tissue sites. The gene pathogens [3] that may help both pathogens and immune expression patterns showed differences between arteries and cells to migrate to target tissues. veins and between large vessel and microvascular ECs. These authors have selected 521 large vessel EC-specific Furthermore, in adults, angiogenesis mainly occurs from the genes and 2521 microvascular EC-specific genes [3, 32]. microvasculature which corresponds to the prefererential This finding suggests that ECs from different locations have expression of angiogenesis-associated genes in microvascular distinct and characteristic expression patterns that persist ECs as compared to large vessels [3]. Similarly, actin- and during in vitro culture [2, 21-24]. myosin-family proteins in the microvascular ECs may be related to their ability to undergo extensive cytoskeletal Recent in situ studies revealed that gene expression patterns of capillaries and venules of brain microvascular remodeling and migration during angiogenesis and infection by pathogens. ECs (BMEC) are similar. But capillaries preferentially express some genes related to solute transport, while venules Although gene expression patterns in cultured cells express an assortment of genes involved in inflammatory clearly reveal the molecular heterogeneity of these ECs, their related tasks [33]. corelation with their in vivo counterparts remains to be defined. 230 Current Immunology Reviews, 2012, Vol. 8, No. 3 Berrich et al.

Endothelia are present in the different classes of arthropod vectors. 26 species, isolated from a wide range of vertebrates, endothelial phenotypes may vary considerably hosts, are recognized, 10 of which are associated to a variety between species in terms of ultrastructure, metabolism, of acute or subacute human diseases (Table 2). B. expression of intracellular as well as cell surface molecules bacilliformis, B. quintana and B. henselae (the two last being [20, 38-39]. close phylogenetically) [42], are responsible for most human infections caused by Bartonellaceae [9, 43]. However, aside Our laboratory has developed nine organospecific feline from causing disease, infection may also result in a chronic, ECs lines, from distinct tissues: eight cell lines from asymptomatic bacteremia [44]. microvasculature and one line from macrovasculature. The cell lines are patented (French patent n° 2010/00094) and Bartonellae have an optimal growth temperature below deposited at the Collection Nationale de Culture de 37°C (B. bacilliformis/26°C, B. henselae and B. Microorganismes (CNCM; Pasteur Institute). quintana/35°C). This temperature could explain why some lesions are localized at body extremities, as well as their Our results revealed intrinsic differences between human survival within an insect vector. and feline ECs concerning kinetics of capillary-like structures formation, persistence of network of pseudo- Clinical Entities Associated with the Most Pathogenic vessels and cAMP production [40, 41]. Thus like all forms of Bartonella for Humans endothelial heterogeneity, interspecies variability may limit the uncritical transferability of findings from animal Species-Related Clinical Entities experiments or animal cell culture systems to the human B. henselae has been identified in 1992 as the main situation or vice versa. etiologic agent of cat scratch disease (CSD), a benign Our data also confirm the existence of significant regional known to occur directly by cat difference of reactivity between ECs belonging to the micro- scratch or bite but cat are suspected to be involved in and macro-vasculature (angiogenesis, wound healing kinetic, some cases [45]. Molecular studies have been found cyclic adenosine monophosphate cAMP and VEGF effective for tracing the feline origin of human infections production). Nevertheless, most studies related to the [46, 47]. mitogenic effects of endothelial growth factors have been In some instances, atypical manifestations in CSD carried out on macrovasculature endothelium, notably patients may develop, including abscessed lymph, Parinaud HUVEC. Thus, in vitro results obtained with HUVEC should oculoglandular syndrome, encephalitis, endocarditis, be confirmed in other types of primary ECs, particularly in hemolytic anemia, hepatosplenomegaly, glomerulonephritis, microvascular ECs because they represent the great majority , relapsing bacteremia, and osteomyelitis [48, 49]. of infected cells in vivo. In addition the precise identity of ECs should be carefully considered for future research and During , B. quintana was a leading cause of therapeutic applications involving manipulations of ECs or as a result of poor sanitary conditions. It is vascular structures. Finally, it is important to correlate the transmitted among humans by the human , results of cell culture models with the clinical situation. Pediculus humanus [50]. The disease is characterized by intraerythrocytic bacteremia, recurrent high-fever episodes, In the next parts of this review, the interactions between headache, and leg pain [50]. the different bacteria for which their interactions with ECs are known to interfere with the clinical course of the B. bacilliformis is responsible for Carrion’s disease [51]. infection and/or with the persistance of the pathogen in the It is transmitted to humans by a nocturnal sandfly, Lutzomyia organism will be presented. Due to the ability of some verrucarum [52]. The disease has two distinct phases, Oroya Bartonella species to stimulate ECs proliferation, an fever and verruga peruana. In immunocompromised important part of this review will be devoted to their individuals, the acute hematic phase (Oroya fever) will relationships with ECs, before considering the interactions occur, characterized by a febrile and severe intravascular between ECs and other bacteria. haemolytic anemia [53]. Patients surviving this acute intraerythrocytic phase have decreased immuno-competency INTERACTIONS BETWEEN THE ENDOTHELIUM and may develop verruga peruana (see infra). BARRIER AND BARTONELLA AND BIOLOGICAL Common Features Associated with Endothelial Cells FUNCTIONS SUBVERSION Involvement Different Bartonella species induce some clinical feature B. bacilliformis, B. henselae and B. quintana can be in accidental host directly related to their interaction with associated in immunocompromised patients to ECs. This interaction between Bartonella and ECs as target vasoproliferative eruptions of the microvascular tissues. in accidental host partly supports the hypothesis of ECs as (BA) is caused by B. henselae or primary niche for Bartonella in reservoir host. B. quintana, particularly in patients infected by human immunodeficiency virus (HIV) [54]. The lesions present as Clinical Manifestations of Bartonella Infections blood filled, ulcerated, crusted, subcutaneous, or dermal nodules. Bacteria are in close association with proliferating Bacteriological Features ECs and infiltration of polymorphonuclear leukocytes and Bartonella spp. are fastidious, hemotropic, gram-negative macrophages [55-57]. Recently Yager et al. (2010) reported a case of BA in immunosuppressed dog induced by B. bacteria that cause a long-lasting bacteremia in their vinsonii subsp. berkhoffii. Skin samples revealed lesions mammalian reservoir host(s) and are mainly transmitted by Vascular Endothelium and Vector Borne Pathogen Interactions Current Immunology Reviews, 2012, Vol. 8, No. 3 231

Table 2. Targeting of the Endothelium by Main Bacteria

Main Recognised Pathophysiology Related to Bacteria Human Diseases Vectors Endothelial Cells

Carrion’s diseases: Oroya fever and verruga Lutzomia peruana Cat-scratch disease, endocarditis, bacillary Inhibition of apoptosis, production of angiomatosis, peliosis hepatis, neuroretinitis, angiogenic factors, activation of Cat meningitis, encephalitis and nimerous other endothelial cells and angiogenesis clinical manifestations induction Trench fever, bacteremia, bacillary Body lice angiomatosis, endocarditis Flea? Endocarditis, lymphadenitis Bartonella clarridgeiae Cat flea Cat-scratch disease in vitro ECs angiogenesis induction Rat flea Endocarditis, neuroretinitis

Bartonella vinsonii subsp. berkhoffii ? Endocarditis, bacteremia, fever, arthralgia, Bartonella vinsonii subsp. arupensis Flea ? neurologic disorders Bartonella koehlerae Cat flea Endocarditis Unknown Conjunctival ulceration, stellar retinitis, Rodents flea neuroretinitis Bartonella waschoensis Flea? Fever, myocarditis in vitro ECs activation and Flea? Unknown angiogenesis induction, production of angiogenic factors Ticks Rocky Mountain

Rickettsia conorii Ticks Mediterranean spotted fever Oxidative damage, ECs activation and Lice apoptosis inhibition Fleas

Oxidative damage, ECs activation and Orientia tsutsugamusbi Chiggers or chiggerborne apoptosis inhibition (or induction?) Human Granulocyte or ECs as a site of replication and Anaplasma phagocytophilum Ticks vascular permeability induction identical to those of human BA providing further evidence estimated that Bartonella species account for 3 to 4% of all that the full range of clinical manifestations of human endocarditis cases [44, 65]. Bartonella infection occurs also in dogs [58]. Similar to BA, (BP) is found in association with Bartonella-Endothelial Cells Interactions proliferating ECs [59] in inner organs such as the liver, spleen, brain, lungs, and bowel [60]. The study of Bartonella-Endothelial cells interactions, mostly in vitro, underlined the different steps (adherence, Verruga peruana may develop as a chronic secondary tissue phase in patients infected by B bacilliformis. During invasion and activation of ECs) leading to angioproliferation and its mechanisms. this phase, the bacteria show tropism for the vascular ECs and cause vasoproliferative eruptions of the skin [61]. The Bartonella-Adherence to Endothelial Cells nodular lesions are primarily localized on limb extremities, the face and neck, though they can be found on the mucosal Bartonella attachment to, and entry into, human lining and, rarely, internal organs. Bacteria are concentrated epithelial cells and ECs has previously been reported [66, in extracellular and interstitial spaces [62, 63]. 67]. B. bacilliformis adhere to and invade ECs, as seen in tissues from verruga lesions and in vitro using primary cell Endocarditis lines [68, 69]. B. henselae and B. quintana can invade Bartonella species including B. henselae, B. quintana, B. nucleated cells in vitro, such as endothelial and epithelial alsatica, B. koehlerae, B. elizabethae, and B. vinsonii subsp cells, by phagocytosis [66, 70]. Invasion of cultured cells berkhoffii and B. v. subsp. arupensis are also responsible for may be preceded by bacteria adherence mediated by the endocarditis indifferent species [64]. Homeless, chronically expression of type-IV-like pili [66] and by the expression of alcoholic individuals and patients with preexisting Outer membrane proteins (Omp) like Bartonella adhesin A valvulopathies are particularly susceptible to the disease. It is 232 Current Immunology Reviews, 2012, Vol. 8, No. 3 Berrich et al.

(BadA) (B. henselae) or its homologue Vomp (B. quintana) The second T4SS of Bartonella, the Trw locus is not [71-75]. required in the vascular endothelium infection but important for establishing erythrocyte infection, as it might already be In contrast to B. quintana and to B. henselae, type IV expressed in ECs to be functional for interactions with secretion systems (T4SS) is not present in B. bacilliformis, so the mechanism of host cell alterations mediated by B. erythrocytes [61]. Moreover, recently Vayssier-Taussat et al. demonstrated that the Trw T4SS of Bartonella mediates bacilliformis must differ significantly [76]. host-specific adhesion to erythrocytes [78]. Bartonella species encode two distinct type IV secretion The endothelial receptors involved in Bartonella systems, VirB-D4 and Trw, which are both required for adhesion may include intercellular adhesion molecule-1 targeting multiple ECs functions and for pathogenesis [61]. The virB-D4 T4SS of these Bartonella is encoded by an (ICAM-1). Interestingly, endothelial adhesion molecules (ICAM-1 and E-selectin) expression is upregulated via NF- operon of 10 genes, virB2-10 and virD4. The expression of B translocation, induced by B. henselae [67]. these proteins is stimulated during in vitro ECs infection [77] and may thus be involved in mediating ECs interaction in In order to adhere to and invade human microvascular both the mammalian reservoir and the incidental human host ECs (HMEC-1), B. bacilliformis engages a family of cell (Fig. 2). receptors called integrins [79]. The primary integrin involved

Fig. (2). Current model of the strategies utilized by Bartonella species to adhere to endothelial cell, invade them and to induce vascular tumor formation. B. bacilliformis has flagella, which facilitates host cell invasion. B. bacilliformis attachment to integrins (directly or indirectly via integrin ligands) leads to their clustering resulting in a signaling cascade involving the protein tyrosine kinase, focal adhesion kinase (FAK) that forms a focal adhesion plaque with paxillin (PAX) and talin (TAL). Activated FAK then activates phosphotidylinsotol 3 kinase (PI3K). Activation of the small GTPases, Rac, Cdc42 and Rho by PI3K results in alterations of the actin network that aide B. bacilliformis internalization. Bartonella henselae can invade human endothelial cells by two distinguishable entry routes: either individually by endocytosis or as large bacterial aggregates by invasome-mediated internalization. Only the latter process is dependent on a functional VirB/VirD4 T4SS and the thereby translocated Bep effector proteins (Bep C, F, and G). BepC and BepF-triggered invasome formation differs from BepG-triggered invasome formation in its requirement for cofilin1, while the Rac1/Scar1/WAVE/Arp2/3 and Cdc42/WASP/Arp2/3 signalling pathways are required in both cases. Like B. henselae, B. quintana can invade human endothelial cells by two distinguishable entry routes: either individually by phagocytosis or as large bacterial clusters. During the phagocytosis process, most bacteria exhibited surface appendages which may mediate specific interactions between the eukaryotic cell and the bacterium. Arp2/3/ Actin- related protein; BadA: Bartonella adhesion A; BCVs: Bartonella-containing vacuoles; BepC: Bartonella effector protein C; BepF: Bartonella effector protein F; BepG: Bartonella effector protein G; Cdc42: Cell division cycle 42; ECM P: Extracellular matrix protein (fibronectin, vitronectin, collagen); Omp43: Outer membrane protein 43; Omp89: Outer membrane protein 89; Pap31: hemin binding protein 31; p38 MAPK: p38 Mitogen-Activated Protein Kinase; Rac1: Ras-related C3 botulinum toxin substrate 1; Rho: ras homolog gene family; Scar1: proteins related to WASP; T4SS: type IV secretion systems; TAAs: trimeric autotransporter adhesin; Vomp: Variably expressed outer membrane proteins; WASP: Wiskott-Aldrich Syndrome Protein; WAVE: WASP-family verprolin-homologous protein. Vascular Endothelium and Vector Borne Pathogen Interactions Current Immunology Reviews, 2012, Vol. 8, No. 3 233 is the fibronectin receptor 51, although the vitronectin triggered by the monocyte chemoattractant protein-1 (MCP- receptor V3 also plays a minor role [79]. 1). During B. henselae infection, it has been shown that MCP-1 was released from ECs and caused chemotaxis of Bartonella-Invasion of Endothelial Cells monocytes and macrophages in vitro [97]. The macrophages/ ECs are invaded by two alternative routes: either by an monocytes recruited by MCP-1 to vasoproliferative lesions [97] would likely propagate chronic inflammation by endocytic uptake of individual bacteria, giving rise to releasing proinflammatory cytokines such as VEGF and Bartonella-containing vacuoles or by the engulfment of interleukin-1beta (IL-1) [87]. clustered bacteria by a unique host cell structure called the invasome [67, 80]. In the case of B. henselae, only the latter Bartonella-Induced Angioproliferation and Vascular process is dependent on a functional VirB/VirD4 T4SS and Tumor Formation the thereby translocated Bep effector proteins [81, 82] (Fig. 2). Both BepC/BepF and BepG-triggered invasome Bartonella are unique among all known bacterial formation inhibit the uptake of individual B. henselae to pathogens in their remarkable capacity to cause proliferation allow bacteria to aggregate on the cellular surface [82]. of microvascular ECs and neovascularization, a process of pathological angiogenesis resulting in the formation of new Invasion is an actin-dependent process and is associated with cytoskeletal re-arrangements induced by Bartonella via capillaries from pre-existing ones [98]. Recent data have indicated that Bartonella can provoke angioproliferation by Rho GTPase signalling [83]. Activated Cdc42 (Cell, division at least two independent mechanisms (Fig. 3): first directly, cycle 42) and Rac (Ras-related C3 botulinum toxin substrate) by triggering proliferation of ECs and inhibiting apoptosis of stimulate the formation of filopodia and lamellipodia leading ECs [99-100]; and second indirectly, by stimulating in a to membrane protrusions, which surround the bacteria and paracrine manner angiogenic factors for the vascular finally engulf them through a phagocytic mechanism [84]. Internal vesicles with single or clumped bacilli are endothelium [87]. colocalized with the Golgi complex in the perinuclear region Bartonella-Induced Angiogenesis - Importance of Bartonella [83]. Induction of the Paracrine Angiogenic Loop Exposure of ECs to B. henselae has been shown to result Angiogenic Effects Observed In Vivo and In Vitro After in aggregation, engulfment, and internalization of the rods, Bartonella Infection forming the invasome structure. This slow invasion process Angiogenesis is a highly orchestrated multistep process (lasting 24 h) is also an actin-dependent mechanism [67]. involving ECs activation, breakdown of the extracellular The bacterial aggregates formed in vitro could correspond to matrix and degradation of basement membranes, ECs the clumps of bacteria observed in close association with the proliferation, EC migration and invasion into the atypically proliferating ECs in BA lesions [59]. More surrounding matrix, and finally the formation of tubular recently Chang et al. showed that succinyl-CoA synthetase structures to build immature vessels later stabilized by tight subunit beta, phage-related protein, and ATP synthase interactions with smooth muscle cells and pericytes [101, subunit alpha might be involved in the B. henselae invasion 102] (Fig. 3). process [85]. Bartonella has been found associated with ECs in Bacterial uptake requires also various host cell kinases bacillary angiomatosis, indicating that the vascular such as protein tyrosine kinases, mitogen-activated protein endothelium represents a target tissue for intra- and kinases (MAPKs) and phosphotidylinositol 3 kinase (PI3K) extracellular colonization in vivo [59]. For example, in cat- that regulate actin organization and internalization [84, 86]. scratch disease, B. henselae has been found in vessel walls Pro-Inflammatory Activation of Endothelial Cells [103]. In angioproliferative lesions, B. henselae is found both within and in clusters surrounding ECs [57, 68, The vasoproliferative diseases caused by Bartonella are 104,105]. Given the close association of bacteria with the indicative of a chronic inflammation, characterized by the proliferated ECs in BA/BP [106], and the fact that antibiotic infiltration of monocytes, macrophages, leukocytes, and treatment leads to regression of those lesions [107], there is polymorphoneutrophils (PMN) [87]. currently a strong belief that Bartonella actively triggers the vasoproliferation [108, 109] and that bacterial colonisation The transcription nuclear factor (NF)-B is considered a of the tumour lesions is critical for tumoral growth. primary regulator of the acute proinflammatory cascade that leads to chronic inflammation. B. henselae itself and B. Relevant to its ability to cause angiogenesis, Bartonella henselae-derived OMPs induce nuclear translocation of the induces migration and proliferation of human ECs in vitro activated NF-B [88] which is involved in the upregulation (Fig. 4) [110, 111]. This angiogenic effect of Bartonella of E-selectin and ICAM-1 in ECs, and in the transcription of species was first reported for B. bacilliformis [112, 113]. It genes associated with the immune response [89-91]. has been demonstrated that B. bacilliformis extracts possess Transendothelial migration of PMN into tissue occurs in an activity that stimulates ECs proliferation up to three times response to chemoattractants such as interleukin (IL)-8 and that of a control [112]. The factor, responsible for this the engagement of platelet-endothelial adhesion molecule-1 proliferation was larger than 12-14 kDa and was thought to (PECAM-1) [88, 92-95]. IL-8 secretion, as shown to occur in be a protein because it was heat sensitive and precipitated the human microvascular endothelial cell line-1 (HMEC-1) with 45% ammonium sulphate [112]. Live bacteria were later in response to B. henselae infection [87, 96], should shown to also have a similar effect [113]. Subsequently, stimulate transendothelial migration of PMN in vivo. The Conley et al. demonstrated in 1994 that the proliferation transformation from acute to chronic inflammation can be stimulating factor of B. henselae was located in the 234 Current Immunology Reviews, 2012, Vol. 8, No. 3 Berrich et al.

Fig. (3). Bartonella host cell interactions and tumour angiogenesis. Bartonella species are capable of infecting a wide variety of different host cells. In particular, they infect endothelial cells, epithelial cells, macrophages and they interact with extracellular matrix. Therefore these bacteria induce production and activation of different bacterial and cellular factor driving angiogenesis. Bartonella species can provoke this angioproliferation by at least two independent mechanisms: directly, by triggering proliferation and inhibiting apoptosis of endothelial cells and indirectly, by stimulating in a paracrine manner angiogenic factors of vascular endothelium by infected macrophages and epithelial cells BadA: Bartonella adhesion A; *BECGF: Bartonella Endothelial Cells Growth Factors; Beps: Bartonella effector proteins; Caspase: Cysteine aspartate specific protease; CXCR2: CXC motif chemiokine receptor (Interleukin-8 receptor); GroEL: heat-shock protein; HIF-1: Hypoxia- inducible factor-1; ICAM-1: Intracellular adhesion molecule 1; IL-1: Interleukin-1; IL-6: Interleukin-6; IL-8: Interleukin-8; MCP-1: Monocyte-macrophage Chemoattractant protein 1; NF-B: Nuclear factor Kappa B; Omp: Outer membrane proteins; PECAM-1: Platelet- endothelial adhesion molecule-1; PMN: polymorphoneutrophils; T4SS: Type IV secretion systems; Tie-1 and tie-2: angiopoietin receptors; TNF-: Tumor Necrosis Factor alpha; VEGF: Vascular Endothelial Growth Factor; VEGFR-2: Vascular Endothelial Growth Factor Receptor 2; Vomp: Variably expressed outer membrane proteins. particulate fraction (prepared by sonication of B. henselae) orchestrates a series of events in addition to endothelial and was trypsin sensitive protein [110]. More recently, it has proliferation (Fig. 3). been suggested that the process through which B. henselae Cats naturally infected with B. henselae display some of the triggers ECs proliferation most likely involves a factor clinical features described for human as endocarditis, but neither released or secreted from the cell, and it has been shown that BA nor BP [116, 118]. Hence, the in vitro effects of infection by direct contact between the bacterium and ECs is not distinct Bartonella strains on human (incidental host) versus necessary to induce ECs proliferation [114, 115]. feline (reservoir host) ECs derived from the macro- and micro- Also, Kirby [99] showed that B. henselae infection of vasculature were compared. Our model revealed intrinsic ECs in an in vitro cord formation assay, in which ECs differences between human micro- and macro-vasculature with overlaid with a type-I collagen gel form a network of regards to angiogenesis kinetics and wound-healing after B. interconnected cells, increases the viability of ECs forming henselae infection [40]. This effect could also be induced by the cords for a longer time than vascular endothelial growth Bartonella culture supernatants. However, no effect was factor (VEGF). In addition, Bartonella infection of ECs in a observed on the feline ECs. These results recapitulate the three-dimensional collagen tube formation assay allowed clinical situation (absence of tumoral lesions in cats vs bacillary cells to invade the matrix, survive in it, and form tubes [99]. angiomatosis in humans) [40]. Therefore, we demonstrated the Taken together, these studies suggest that Bartonella crucial role of the species and tissue of origin of ECs [40]. Vascular Endothelium and Vector Borne Pathogen Interactions Current Immunology Reviews, 2012, Vol. 8, No. 3 235

Fig. (4). Capillary-like-induced formation of Human Skin Microvascular Endothelial Cells (HSkMEC) by Bartonella henselae infection [40]. HSkMEC infection by B. henselae (with a multiplicity of infection (MOI) of 100 bacteria per cell) induces capillary-like structure formation of HSkMEC as compared to uninfected cells. Pictures have been taken 20 h after seeding HSkMEC on MatrigelTM. Original magnification: 50x. B. henselae: reference strain Houston-1 ATCC 49882.

Mechanims of VEGF Production and its Biological VEGF at levels supporting HUVEC proliferation [122]. Consequences Recent data showed that B. henselae infection abrogated VEGF induced proliferation and wound closure of ECs VEGF was proved to be critical and specific for blood monolayers as well as the capillary-like sprouting of ECs vessel formation [119]. It plays a fundamental role in spheroids [123]. But this study was carried out with pathological angiogenesis [119] and is a highly specific HUVEC. Using our human vs feline ECs model [40, 41], we proliferative factor for ECs [120]. This factor also seems recently found that B. henselae triggers VEGF production of involved in Bartonella-induced angiogenesis. Both increased VEGF production by patients infected by Bartonella and human Skin Microvascular ECs but not of HUVEC or of feline macro- or micro-vascular ECs (Berrich et al, increased Vascular Endothelial Growth Factors Receptors unpuplished data). Our results may explain the absence of (VEGFR) expression at the surface of the infected ECs have reactivity to Bartonella infection in cats as compared to been described and could require multiple factors for humans and strongly suggest that an autocrine secretion vasoproliferation and angiogenesis in vivo. could play a certain role, in addition to the paracrine loop. For instance, studies of clinical specimens of verruga In contrast to ECs, activated inflammatory cells are peruana show that the primary source of VEGF is the generally considered high VEGF producers [87]. These data overlying epidermis [121]. High-level expression of are in favor of a paracrine angiogenic loop in which B. angiopoietin-2 and VEGF receptors (VEGFR-1 and VEGFR- henselae can stimulate an autocrine loop of VEGF-mediated 2) was also observed in the endothelium of verruga peruana endothelial proliferation [87, 124]. [121]. Moreover, infection of cultured endothelium with B. bacilliformis resulted in induction of angiopoetin-2 in vitro. As histological examination of BA lesions shows the In addition, prominent expression of angiopoietin receptors, infiltration of polymorphonuclear leukocytes and the tie-1 and tie-2, was observed in ECs of verruga peruana by in presence of macrophages [55-57], macrophages may also situ hybridization [121]. This finding points to a novel form represent target cells for Bartonella. The interaction between of angiogenesis, in which proliferation of endothelium may these professional mononuclear phagocytic cells and bacteria be due to autocrine loops of angiopoietin-2 and tie-2 may trigger the production of inflammatory or angiogenic (angiopoietin receptor), with paracrine contributions of cytokines and growth factors with autocrine or paracrine VEGF produced outside the infected endothelium [121]. functions [87]. B. henselae was reported to be effectively phagocytosed by J774 cells and to survive within this mouse Moreover, immunohistochemistry for VEGF in macrophage cell line for at least several hours [125]. specimens from patients with BA or BP caused by B. Moreover, macrophages are thought to play a central role in henselae revealed increased VEGF expression in vivo [122]. There is a debate about the autocrine and/or paracrine source the modulation of angiogenesis by means of their secreted VEGF [87, 120]. Several other macrophage-derived of VEGF that links to VEGFR, but according to Maeno et al, mediators of angiogenesis have been described, including ECs did not produce VEGF but were able to proliferate in IL-8, IL-6, IL-1, and tumor necrosis factor alpha (TNF-) response to B. henselae infection [114]. A further study by [126, 127]. Both VEGF and IL-1 are released from the Kempf et al. showed the importance of VEGF in B. human macrophage cell line THP-1 in response to infection henselae-mediated ECs proliferation [122]. However, ECs are not the major cell type producing VEGF [122]. B. with B. henselae. Importantly, a conditioned culture medium resulting from the cultivation of macrophages in the vicinity henselae infection of Ea.hy 926 cells triggered secretion of 236 Current Immunology Reviews, 2012, Vol. 8, No. 3 Berrich et al. of Bartonella was able to trigger proliferation of HMEC-1 with that, nuclear HIF-1 was detected in Bartonella-infected (Human Microvascular Endothelial Cell line) which may cells in vitro and in vivo in BA patient samples, which contribute in a paracrine manner to the proliferation of ECs suggests that it could be a key factor driving to the [87]. Pretreatment of macrophages with cytochalasin D, a vasculoproliferative disorder [122, 133]. phagocytosis inhibitor, yielded comparable results, Other Mediators of Bartonella-Induced Angiogenesis suggesting that bacterium-cell attachment is sufficient for VEGF and IL-1 induction [87]. It is unlikely that VEGF was the sole mediator of Bartonella-induced angiogenesis. Kempf et al. showed that PolyMorphoNuclear cells (PMNs) are also capable of the administration of VEGF-neutralising antibodies reduced secreting VEGF in response to bacterial infection, i.e. endothelial proliferation by only 50% [122], suggesting that Streptococcus pneumoniae [128]. These cells represent other factors known to play essential roles in angiogenesis another major constituent of the mixed inflammatory are involved, such as basic fibroblast growth factors (bFGF), infiltrates of Bartonella-triggered bacillary angiomatosis. placenta growth factor (PIGF), angiopoietins, ephrins [119] However, no study has explored how Bartonella may or yet unknown angiogenic factors. The presence of vascular stimulate VEGF secretion by PMN. In the present state, it lesions in the maternal placenta of mice infected cannot be totally excluded that Bartonella infections of host experimentally with B. birtlesii [134], suggest that PIGF cells induce a mitogenic activation or lead to a stress might be involved in the genesis of the reproductive response of epithelial cells, giving rise to a secondary disorders observed in infected mice. production of vasculoproliferative cytokines [121, 122]. Also, a recent study revealed that the VirB/VirD4 type Thus, a paracrine loop should play a much more IV secretion system and a subset of its translocated important role than an autocrine loop. A bacterial 2-step Bartonella effector proteins (Beps) profoundly modulate B. pathogenicity strategy would take place, in which B. henselae-induced sprouting activity [102]. BepA, known to henselae might trigger VEGF production in host cells to protect ECs from apoptosis, strongly promoted sprout speed up endothelial proliferation, which in turn would formation. In contrast, BepG, triggering cytoskeletal promote bacterial growth [122]. Nevertheless, the majority rearrangements, potently inhibited sprouting [102]. Hence, of studies have been carried out on macrovascular ECs (i.e. an in vitro model of Bartonella induced angiogenesis HUVEC). revealed distinct and opposing activities of the type IV Concurrently to this paracrine loop in which the infected secretion system and of its effector proteins [102]. macrophage effector cells secrete ECs mitogens [87], the B. henselae is not typically found inside ECs during BA infected ECs may upregulate expression and production of [104]. There have been reports of B. bacilliformis found pro-angiogenic proteins such as chemokines, adhesion inside ECs in vivo; however, many more bacteria were found molecules [88], and cytokines [87, 122]. During B. henselae outside the cells [68]. Thus, studies focusing on the infection, monocyte-macrophage chemoattractant protein 1 interactions between ECs and extracellular Bartonella may (MCP-1) released from ECs can cause chemotaxis of be more physiologically relevant than studies on the monocytes and macrophages to the site of infection, thereby intracellular effects of these bacteria. Therefore, it was promoting the potential paracrine loop by recruiting the demonstrated that cell-free B. henselae-conditioned medium effector cells or can also directly promote angiogenesis of (BCM) enhanced CXCL8 production and HUVEC ECs [97, 129]. proliferation in a Ca2+-dependent manner [115]. When the Importantly, and whatever the way it is produced, VEGF proteins from the B. henselae lysate and the cell-free B. not only induces endothelial proliferation but also cell henselae supernatants are compared, there are protein bands migration through several pathways that include the unique to the supernatants, suggesting that these may be activation of a small GTP-ase RhoA, which is associated secreted proteins [115]. with phosphorylation of myosin light chain [130]. This may Moreover, BCM contains BadA and GroEL a heat-shock explain why Bartonella altered spatial organisation within protein, both of which are important during B. henselae the monolayer and changes in cell morphology due to infection of ECs [72, 135]. In addition, recent report cytoskeleton re-organisation [111, 131]. In addition, the proposes the molecular chaperone GroEL from B. impaired migratory ability of Bartonella-infected ECs might bacilliformis as a potential vasoproliferative candidate [135]. result from the formation of thick robust stress fibres, However, the GroEL from B. henselae present in the lysate probably via the activation of RhoA [83, 131]. Two studies was mitogenic at a sixfold lower rate than GroEL present in found that B. henselae induces VEGF secretion from the lysate from B. bacilliformis [115]. Additional carcinoma [122] and monocytic [87] cell lines, respectively. experimental evidence is required to unequivocally The inflammatory infiltrates could be a mitogen sources in demonstrate the molecular role of GroEL in vascular vivo, because B. henselae-infected macrophages release proliferation. some angiogenic factors [87]. Bartonella Inhibition of Endothelial Cell Apoptosis Some authors have also investigated the mechanisms by which VEGF production is induced after Bartonella Apoptosis is a process of cellular suicide that acts as a infection. Hypoxia-inducible factor-1 (HIF-1) is known as a homeostatic mechanism for controlling cell proliferation, key transcription factor for the induction of VEGF [132]. because excessive death may lead to compromised Bartonella adhesion A (BadA) mediated the adherence of B. development or degenerative disease, while the lack of cell henselae to ECs and triggered the activation of the death could result in proliferative disorders [136]. It is proangiogenic program through HIF-1 [72]. In accordance considered a primary defense against infections [137]. In the Vascular Endothelium and Vector Borne Pathogen Interactions Current Immunology Reviews, 2012, Vol. 8, No. 3 237 case of Bartonella, the formation of vascular tumors in exception of B. bacilliformis, suggests that the re-infection immunocompromised patients was shown to be linked to the waves are due to the liberation of the bacteria from a distant inhibition of apoptosis of infected ECs [100]. sanctuary site [131]. What is clear is that Bartonella can colonize secondary foci at considerable distances from the It has been showed that Bartonella suppresses both early and late events in apoptosis, namely caspase activation and primary site of infection, and there is a preference for highly vascularized tissues like heart valves, liver and spleen (B. DNA fragmentation, respectively [100]. The antiapoptotic quintana and B. henselae), or vascular bed of the skin (B. mechanism, appears to involve the specifically inhibition of bacilliformis) [143]. the caspases 3 and 8 [100]. As vascular endothelium interacts with circulating blood In vitro B. quintana first stimulates ECs apoptosis as early as 6h post infection inducing a very early cells in vivo, ECs could be involved in pathogenesis and dissemination of the organisms. Circumstantial evidence overexpression of caspase 8 and Apaf-1, an increasing suggests that they represent a major constituent of this mRNA production of TNF-, IL-8, and E-selectin and an primary niche [144,145]. activation of p38 MAPK (Mitogen-Activated Protein Kinase) and of Stress Activated Protein Kinase/ c-Jun-N- In a rat model of green fluorescent protein (GFP)-tagged terminal kinase (SAPK/JNK), with bacterial clusters strain of B. tribocorum infection, the presence of periodic appearing at the cellular surface of the HUVEC [138]. erythrocyte infection waves has been precisely described However, at 8 to 24 hours postinfection, B. quintana was [146]. Following intravenous injection, the bacterial internalized and inhibited proapoptotic signals such as p38 inoculum is rapidly cleared from the bloodstream. MAPK and SAPK/JNK while inducing antiapoptotic signals Approximately five days post-inoculation, large numbers of such as the expression of the bcl-2 gene and the increase of bacteria are released from the primary niche into the the bcl-2 kinase active form [139]. bloodstream. The primary niche was suspected to be the ECs [146]. The balance between the two Bcl-2 family members, bcl- 2 (antiapoptotic) and bax (apoptotic) is important for ECs Most Bartonella species do not appear to interact survival or apoptosis [140]. IL-8 induces an increase in Bcl-2 strongly with erythrocytes in vitro [144, 147-149], expression and a decrease in bax expression, which most suggesting that the colonization of the elusive primary niche likely favors survival over apoptosis in ECs. In the presence is required to gain competence for erythrocyte interaction of B. henselae, the expression of bax is decreased and the [61]. The exception is B. bacilliformis, which infects most of expression of bcl-2 is increased. These data revealed a the erythrocyte population during natural infection in human possible role for IL-8 in the prevention of apoptosis [96]. In and which also binds to and invades human erythrocytes in addition, Chang et al. [85] demonstrated that B. henselae- vitro [53, 150-153]. induced cell proliferation involved the mitochondria intrinsic Beside skin, bone is the second most frequent site of apoptotic pathway. bacillary angiomatosis [154]. Since then, a number of hints A recent study showed that the inhibition of apoptotic suggest that the primary niche might instead be located in the cell death by B. henselae requires a functional VirB/VirD4 bone marrow. Candidates are mainly the cells that are issued by T4SS [141]. Bartonella effector proteins (Bep), translocated differentiation of the haemangioblast, the common precursor of into the host cell via the VirB/VirD4 T4SS, are necessary both erythrocytes and ECs. These include mainly angioblasts and sufficient to inhibit ECs apoptosis [142]. During and erythroblasts. Erythroblasts express VEGF [155], which is HUVEC-infection, the anti-apoptotic activity appeared to be also known to enhance Bartonella replication [122]. This limited to BepA orthologs of B. henselae and B. quintana hypothesis is supported by recent studies in which cultivation and correlated with elevated levels of intracellular cyclic and immunofluorescence detection of B. quintana in bone adenosine monophosphate (cAMP) [142]. marrow-derived erythroblasts of homeless people was reported [156, 157]. Such colonization of erythropoietic lineage would However, these studies are exclusively based on the use of HUVEC. Those cells are very different from protect B. quintana from the host immune response and explain the periodic bacteraemic relapses. microvasculature-derived ECs involved in BA and BP. Using our human macrovascular and microvascular cellular Consistent with this suggestion, recent studies elucidated model, we showed that HSkMEC (Human Skin whether human CD34+ hematopoietic progenitor cells Microvasscular ECs) did not produce high levels of cAMP (HPCs) internalize B. henselae and may serve as a potential as opposed to HUVEC [40]. niche of the pathogen [149]. By means of confocal microscopy and via gentamicin protection assays, B. Given that antiapoptotic activity alone cannot account for henselae was shown not to infect human erythrocytes. In the increase in cell number observed for Bartonella-triggered contrast, B. henselae was able to infect HPCs to a similar vascular proliferation in vivo, it seems unlikely that apoptosis extent as ECs. These data support the hypothesis that was the sole mediator of Bartonella-induced angiogenesis. erythrocytes do not function as the primary target in B. henselae infections. These observations might be explained Endothelial Cells as Primary Niches for Bartonella spp. by the fact that B. henselae binds to host cells via beta-1 integrins [72] and that beta-1 integrins are expressed on A common feature between different Bartonella species HPCs or reticulocytes and on ECs but not on mature is their haemotropic lifestyle. Because vectors do not directly erythrocytes [72, 149, 158]. The extent of proliferation of B. tap blood vessels, an intermediate tissue may mediate henselae-infected HPCs was, however, significantly lower infection of blood cells. The fact that Bartonella parasitise erythrocytes without leading to haemolysis, with the compared with uninfected control cells [149]. 238 Current Immunology Reviews, 2012, Vol. 8, No. 3 Berrich et al.

The marked tropism of Bartonella spp. for ECs [61], the and/or entry into host cells, including ECs, are rOmpA & B. proximity of ECs to the bloodstream, the fact that Bartonella They belong to a family of autotransporters called Rickettsial species remain undetectable for several days upon surface cell antigens (Sca). At least 17 predicted sca genes experimental infection [134, 146, 159], and that these have been identified [162]. rOmpB (Sca5) is present in both pathogens are detected in the erythrocytes during the course SFG and TG Rickettsia whereas rOmpA (Sca0) is absent in of infection [146], suggest that ECs might be the primary TG Rickettsia [163]. The host cell receptor of OmpB has niche. been identified as Ku70 [164], a signal transduction protein that is as nuclear DNA dependent protein kinase, also These observations might suggest a newly described present in the cytoplasm and on the plasma membrane of pathogenicity strategy for bacteria, in which the infection of ECs. The involvement of other bacterial [162] and cell primary niche cells is required to gain competence for erythrocyte infection [61], resulting in the spread of the receptors is strongly suggested by recent data relating to R. conorii [161, 162]. Other Sca molecules (at least 17 bacteria and for chronic infections. The unique adaptation to predicted sca genes have been identified [162]), could play colonize this intracellular niche increases the chances for an important role in bacterial adherence to host cells, in such transmission to other susceptible hosts via blood-sucking a way that attachment to mammalian cells is uncoupled from ectoparasites. the entry process [165]. Sca1, which is present in almost all Rickettsia, has recently been found to promote adherence to INTERACTIONS BETWEEN OTHER BACTERIA non-phagocytic mammalian cells, including ECs, but not cell AND ENDOTHELIAL CELLS invasion [165]. Whatever the molecules involved in bacterial-cells attachment, one key and common feature of Rickettsia-Endothelial Cells Interactions ECs response to intracellular agents, particularly Rickettsia Rickettsia Clinical Associations belonging to SFG, is the stimulation of host cell actin polymerization, leading to expression of prominent actin Bacteria belonging to the genus Rickettsia are arthropod stress fibers [4]. The entry is very rapid (a few minutes) after borne bacteria that are strictly intracellular. It has been cell contact, and SFG bacteria quickly escape into the recently suggested that the most recent common ancestor of cytoplasm of mammalian cells [161]. The rOmpB-dependent the Rickettsiales was probably already adapted to arthropods mechanism of Rickettsial internalization involves and that the infection of mammals including humans cholesterol-rich micro-domains containing Ku70 and a occurred later in their evolution [160]. cytoskeletal actin-mediated zipper mechanism of Rickettsia induce severe febrile diseases with systemic endocytosis. multi-organ involvement (Table 2), which are frequently According to Chan et al. (2009) [166], the interaction fatal even in young immunocompetent individuals. Two between rOmpB and Ku70 is sufficient in the case of R. main groups can be distinguished within Rickettsia: (a) the conori to recruit the actin cytoskeleton via multiple spotted fever group (SFG) Rickettsia which include R. pathways, including the involvement of p-Tyrosine kinases rickettsii, the agent of Rocky Mountain spotted fever (pTK), PI-3 kinase, the GTP-binding protein Cdc42. In the (RMSF) and , the agent of Mediterranean R. conori model, this process is at least partly dependent spotted fever (MSF); and (b) the typhus group (TG) upon the activity of c-Cbl, an ubiquitin ligase that is also rickettsia, which include R. prowazekii, the agent of suspected to play the role of a signaling molecule. These epidemic typhus and Rickettsia typhi, the agent of murine or pathways drive ultimately to the activation of the actin endemic typhus. R. rickettsii and R. prowazekii are nucleating complex, Arp2/3, which results in bacterial particularly dangerous pathogens for humans within this endocytosis by ECs and other non phacocytic mammalian bacterial group. R. rickettsii causes up to 20% mortality cells [163]. rates, and R. prowazekii 10-60%, if no antibiotic treatment is In the case of R. Rickettsi, another SFG (Fig. 5) a recent applied. The typical symptoms induced by these bacteria are study evidenced that their internalization into ECs involves related to their affinity to the endothelium of small and medium-sized blood vessels, which results in the “Rickettsial signalling through the p38 module of MAPK [167]. Viable bacteria are necessary for inducing p38 phosphorylation and vasculitis”, characterized by the association of different activation, which in turns stimulates actin polymerization, types of vascular troubles, i.e. damage in blood vessels, leading to actin filaments close to those of filopodia and increased permeability and vascular inflammation and allowing this Rickettsia to spread directionally within cells dysfunction (Fig. 5) [161]. As these alterations can affect and between cells via actin mobility [4]. Despite this effect vessels located in many organs due to widespread infection, the clinical signs can be very polymorphic: from of Rickettsia on actin polymerization, no disruption of the stability of the cytoskeleton and of the inter-endothelial maculopapular and petechial rash in a majority of patients to adherens junctions is observed. In addition, a recent study encephalitis, pulmonary oedema, acute renal failure due to has shown that an other Sca, Sca2, which is present in most hypovolemic hypotension, multiple organ failure. SFG Rickettsia but not in TG [163] could play a role in intra- Endothelium Barrier Breaching by Rickettsia and inter-cellular motility, by polymerizing actin monomers and elongating filaments in a profiling-dependent manner Rickettsia-Invasion and Intracellular Colonization of [168]. Endothelial Cells Conversely, Rickettsia belonging to TG display no or very The two first Rickettsial outer membrane proteins short actin tails. As a consequence, they tend to accumulate in (rOmp) that have been involved in the bacterial adherence the cytoplasm ( 5-8 times more than SFG) until cell lysis. Vascular Endothelium and Vector Borne Pathogen Interactions Current Immunology Reviews, 2012, Vol. 8, No. 3 239

Fig. (5). Effects of R. rickettsii on endothelial cells and clinical consequences. R. rickettsii possesses both OmpA & OmpB (that links to Ku70) receptors, but other receptors are probably involved. The interaction with Ku70 results in an activation of MAP kinases, particularly 38-MAPK, which cascade of activation drives to the internalization of R. rickettsii, through cell actin polymerization. 38-MAPK is also involved in the stimulation of MCP-A and of the coagulation cascade, but not in the process of activation of NF-B. This nuclear factor plays a central role in the induction of vascular permeability and oedema, through the transcription of different effectors which stimulate prostaglandin secretion (but PGE2 production is not activated in micro-vascular ECs). This is a complex pathway as HO-1 reduces the oxidative stress but also stimulates COX-2 secretion. NF-B also inhibits endothelial apoptosis, thus contributing to intracellular survival of R. rickettsii, and stimulates the inflammatory and coagulation cascades. CCL2: Chemokine ligand 2; Cxcl: CXC chemokine ligand; COX-2: Cyclooxygenase-2; HO-1: Heme oxygenase 1; IL-1: Interleukin-1; IL-6: Interleukin-6; IL-8: Interleukin-8; NF-B: Nuclear factor Kappa B; MCP-1: Monocyte-macrophage Chemoattractant protein 1; NO: Nitric oxide; OmpA: Outer membrane protein A; OmpB: Outer membrane protein B; p38 MAPK: p38 Mitogen-Activated Protein Kinase; Pal-1: Phe ammonia lyase 1; PGE2: Prostaglandin E2; PGi2: Prostacyclin; TF: Tissue Factor; VCAM-1: Vascular cell adhesion molecule-1.

Endothelial Cells Injury by Rickettsia NFB, a Key Factor for Rickettsia-Endothelial Cells Activation ECs injury, which is associated to extensive membrane damage and eventual cell death, is presently known to be Permeability is increased and the activation of an induced not simply by cell infection and Rickettsial exit but inflammatory response gives rise, in the case of SFG also by immune CTL induction of apoptotic death of Rickettsia, to transcription factors including NF-B, and infected cells [169] and by “endothelial activation” induced consequent expression of cytokines, chemokines, and adhesion by intracellular Rickettsial infection, which results in the molecules. acquisition of a proinflammatory and procoagulant Among the genes that are transcriptionally up-regulated phenotype. early after in vitro infection (e.g. genes encoding adhesion Rickettsia-Infected Endothelial Cells and the Immune molecules like E-selectin and VCAM-1, von Willebrand factor, System IL-1, IL-6, IL-8, MCP-1, PAI-1, heme oxygenase-1(HO-1), cyclooxygenase-2 (COX-2), tissue factor (TF), CCL-2, CXCL, ECs particularly those belonging to the micro-circulation, prostanoids via COX-2, and NO [170]), a majority have NF-B have very close contacts with T lymphocytes, which allows binding sites in their promoter regions [161]. In vitro, NF-B them, through the expression of class I and II MHC antigens, to play a major role in antigenic presentation in humans. It is also inhibits endothelial apoptosis, which has important consequences on the outcome of the infection [171]. known that Nitric Oxide (NO) is cytokine-directed and drives to Rickettsial killing, providing a major host defense mechanism, In vitro, Human ECs infected with R. rickettsii display NF- through intracellular autophagy. In vivo, in individuals affected B activation as early as 1.5 h postinfection with an early, by MSF and/or , increased concentrations transient peak at 3 h, followed by a later, sustained phase at 18- of IL-6, IL-8, IL-10, IFN-, TNF-, RANTES, INOSs and 24 h. indoeamine 2,3-dioxygenase are associated with ECs and In addition, R. rickettsii is also able to interact directly with lymphocyte activation in cutaneous lesions [4]. the inactive form of NF-B present in ECs cytoplasm [172].

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Direct activation of NF-B by intracytoplasmic R. rickettsii adherens junctions [180]. Increased transmigration of apparently depends on a yet unidentified bacterial protease leukocytes occurs, most likely through the exacerbated activity. inflammatory response [181]. The induction of proinflammatory cytokines IL-1, IFN- and TNF-, Rickettsia-Resistance to the Oxidative Stress exacerbate the effects of Rickettsia, which suggests that a Following infection with R. rickettsii, the infections combination of bacterial and host components, including the culminates in widespread dilatation of intracellular role of the immune response in addition to that of the membranes, loss of osmoregulatory control, striking changes prostaglandins, contribute to such enhanced permeability. in the structure of the cytoplasm and the cytoskeleton, and Animal models of Rickettsial infection have been cell lysis at 5 or 6 days postinfection. developed, using intravenous R. typhi or R. conorii infection One important feature associated with these dramatic of C3H/HeN mice. For both bacteria, ECs represent targets changes is the oxidative stress occurring during intracellular in vivo and express various chemokines in response to infection, resulting in the accumulation of intracellular infection (CCL2, CCL3, CCL4, CCL12, CX3CL1, CXCL9, peroxides and superoxide radicals, higher amounts of and CXCL10) [182, 183]. extracellular H2O2, and alterations in the activities of important antioxidant enzymes [173, 174]. But a beneficial Interferons have also been demonstrated to play an important role in Rickettsial pathogenesis. IFN- and IFN- effet of antioxidant compound -lipoic acid in R. rickettsii- were found to have inhibitory activity on the growth of infected endothelium was observed in vitro. Moreover, in R. prowazekii [184]. The ability of host ECs to kill Rickettsia vivo, in a mouse model of RMSF, activation of important depends on the stimulatory effects of IFN-, IL-1, antioxidant enzymes in host tissues is correlated with RANTES and TNF- [169, 185, 186]. Eventually, IFN- Rickettsial titers in target host tissues, suggesting a protecting effect for different molecules, like -lipoic acid, appeared to play an important protective role during infection with different Rickettsia in vivo studies using HO-1 and ferritin, which is a potent antioxidant and anti- animal models [169, 185, 186]. This role appears to be apoptotic molecule [175]. The heme-HO system contributes associated with Natural Killer (NK) cells, at least in the case to reduction of oxidative stress, of vascular constriction, of of R. conorii [161]. inflammation, decrease in smooth muscle cell proliferation and inhibition of apoptosis. The heme-HO system also Action on Endothelial Cells Apoptosis, a Way to Rickettsia increases the expression of vascular COX-2 which in turns Survive Intracellularly induces increased levels of prostaglandin secretion [175]. For intracellular bacteria, apoptosis can be considered as The prostaglandins PGE2 and PGI2 cause vascular detrimental for bacterial survival. Thus, it is not surprising permeability and edema, considered to be cardinal features that the majority of these bacteria have developed anti- of acute inflammation during Rickettsial infections [161]. apoptotic mechanisms. In the case of ECs, apoptotic cells detach from the vessel basement membrane and are rapidly Inflammation and Coagulation, Two Effects of “Endothelial cleared by phagocytes. ECs apoptosis is indeed not favorable Activation” to Rickettsial survival, which has suggested to different In patients, an activation of the coagulation cascade authors that inhibition of apoptosis could represent a strategy occurs. Hemostasis and adhesion to infected endothelium of survival for these bacteria. In accordance with this consume platelets, but significant hemorrhage and hypothesis, it has been shown [171] that the anti-apoptotic disseminated intravascular coagulation (DIC) are rare as functions of NF-B are essential for ECs survival during R. non-occlusive hemostatic plugs are present only in foci of rickettsii infection. NF-B prevents the activation of endothelial destruction [4]. It has also been suggested that caspase-8 and -9 mediated pathways, thus preventing the DIC is rare in rickettsial infections as compared to sepsis, activation of downstream effector caspases -3, -6, or -7. because Rickettsiae target the arterioles and venules, rather Thus, NF-B protects ECs against infection-induced than endotoxin mediated damage to capillary endothelial apoptotic death. cells [176]. Indeed, a recent in vitro study has demonstrated A recent study [187] has shown that R. rickettsii infection that macrovascular but not microvascular ECs present protects Human microvascular ECs against staurosporine- increased activity of COX-2, which in turns results in induced apoptosis by a cIAP2-independent mechanism, increased amounts of PGF2 in macrovascular ECs only supporting the hypothesis that intracellular Rickettsia could [177]. But sepsis mediated endothelial injury is not considered in this review. participate in the regulation of the programmed host-cell death by more subtle mechanisms. Among the genes As previously mentioned, activation of NF-B and implicated in the regulation of the apoptotic/anti-apoptotic stimulation of MAPKs, particularly p38 MAPK, the process, 14 genes were found significantly up-regulated downstream effector component of a stress-activated MAPK [187]. cascade, results in increased secretion of different proinflammatory effectors, like IL-1, IL-6, IL-8 and MCP-1 The modulation of the expression of both the anti- apoptotic factors (up-regulation) and some of the apoptotic in host ECs infected by SFG Rickettsia [167, 178, 179]. In factors (reduction) could contribute strongly to the anti- the case of R. prowazekii (TG Rickettsia), infected cells apoptotic effect observed with Rickettsia [187], display increased prostaglandin secretion, resulting in counterbalancing efficiently the bacterial and cellular factors enhanced permeability, associated with the disruption of that could promote ECs apoptosis.

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Orientia-Endothelial Cells Interactions through the activation of NF-B, this effect seems absent in the cases of O. tsutsugamushi [4]. This bacterium could even Orientia Clinical Associations trigger an apoptotic effect as a consequence of diminished focal adhesions associated with the infection. Nevertheless, The genus Orientia belongs to the same family than the genus Rickettsia. O. tsutsugamushi is the agent of a human this effect has been observed only in heavily infected cells. disease named tsutsugamushi disease, scrub typhus or To date, contradictory results have been observed by chiggerborne rickettsiosis (Table 2). It is observed in Asia, different authors when studying the interactions between northern Australia, and adjacent islands of the Pacific and these bacteria and ECs. In the majority of in vitro models Indian Oceans and is transmitted by larval trombiculid developed, O. tsutsugamushi infection induces or increases (chiggers). O. tsutsugamushi also infects ECs. According to the level of expression of CXCL8, CCL2 and CCL5. This recent studies, the concentration of TNF- may predict the effect has been observed with a human dermal microvascular severity of tsutsugamushi disease in the acute infectious endothelial cell line, with an endothelial-like cell line and phase, as it was significantly elevated in 8/9 patients [188]. with HUVEC, associated with increased production of the The levels of IL-10 and IL-12p40 were also found elevated transcription factor Activator Protein-1 (AP-1) and could be in all patients in the acute phase, above the normal upper reproduced using heat-inactivated O. tsutsugamushi [4]. In limits, and IFN- levels were elevated in 44% of patients the case of the two first cell lines, this effect did not need the [188]. activation of NF-B. Nevertheless, in HUVEC cells, DNA- binding activity of both AP-1 and NF-B was activated in Endothelium Barrier Breaching by Orientia HUVEC, but AP-1 was more obviously involved in MCP-1 activation. Like for Rickettsia, a transcriptional control of Orientia-Invasion and Intracellular Colonization of MCP-1 by the MAPK pathways has been demonstrated. In Endothelial Cells the case of O. tsutsugamushi, both p38 MAPK and Like Rickettsia, O. tsutsugamushi, use ECs as their main MAPK/ERK (extracellular signal-regulated kinase) kinase targets of intracellular infection. Recent studies indicate that (MEK) are involved [7]. adherence to and invasion of eukaryotic host cells by O. tsutsugamushi occur through integrin-mediated signalling, Anaplasma-Endothelial Cells Interactions using a multistep process [189]. A major outer membrane protein, TSA56, a 56 kDa type-specific antigen, binds to Anaplasma Clinical Associations fibronectin, which facilitates bacterial entry into host cells, potentially via interactions with integrins [190]. Indeed, the The genus Anaplasma belongs to the Anaplasmataceae bacteria colocalize with integrin 51, inducing the family, the second family in the Rickettsiales order. As such, activation of several integrin signaling effectors such as Src it is an obligate intracellular tick-borne bacterium. Two tyrosine kinases and RhoA GTPase, and the recruitment of species of this genus, A. phagocytophilum and A. marginale signalling adaptors, such as talin and paxillin. Actin are known to have interactions with ECs. As other tick-borne reorganization and membrane protrusions at the sites of bacteria, A. phagocytophilum and A. marginale infect blood infection of nonphagocytic host cells are also observed. cells, granulocytes and erytrocytes respectively [193]. Apart from TSA56, Sca molecules could contribute to According to the blood cells that are infected, the clinical bacterial-cell adherence; like for Rickettsia 6 sca genes have consequences associated with these two species are different. been identified so far in the of O. tsutsugamushi A. phagocytophilum is isolated from humans, equines, [191]. ruminants, rodents, carnivores. It causes the Human Granulocyte Ehrlichiosis/Anaplasmosis (HGE/A), Orientia Factors Involved in Pathogenesis characterized by the induction of a nonspecific febrile illness The inflammatory effect induced by O. tsutsugamushi on with leukopenia, , and mild liver injury ECs begins to be better understood. The recent publication (Table 2). In cattle and sheep, A. phagocytophilum causes the from Cho et al. [192] has shown, using a human endothelial pasture fever or tick-borne fever, which is characterized by cell line, ECV304, that a strong induction of CCL5, CCL17, high fever, reduced milk yield, leucopenia, abortion and IL-1, IL-6, IL-8, IL-10, IL-15, TNF- and TNF- was reduced fertility. In both species, there is a reduction of observed after O. tsutsugamushi infection. In addition, immuncompetence in infected patients. NOD1 (Nucleotide-binding oligomerization domain) A. marginale infection in cattle is characterized by severe pathway activation was associated with significant increase anemia associated with intra-erythrocytic , of IL-32, a proinflammatory cytokine that is constitutively resulting in fever, depression, and weakness, and the disease expressed in ECs and that is increasingly suspected to induce induces economic losses. Infected cattle that recover remain IL-1, IL-1, IL-6, and TNF- via the NF-B and p38 reservoirs of bacteria [194]. MAPK pathways. NOD1 knockdown resulted in reduced IL- 1, IL-6, IL-8, and ICAM-1 expression in O. tsutsugamushi- Anaplasma and Endothelial Cells infected ECV304 cells. Consequently, the authors hypothesize that NOD1 pathway may act on cytokine release ECs are suspected to represent either a site for initial in ECs as a modulator of the inflammation caused by O. replication after tick-borne transmission or the tissue tsutsugamushi infection. reservoir for Anaplasma spp. during persistent infection, as neither infected granulocytes nor infected erythrocytes can Conversely, little reliable information is available assume this role [193]. No effect on apoptosis has been concerning other effects of these bacteria on ECs. In contrast to Rickettsia that appear to induce an anti-apoptotic effect 242 Current Immunology Reviews, 2012, Vol. 8, No. 3 Berrich et al. described to date for the bacteria belonging to this genus and the authors to hypothesise that ECs could play an important to its family. role in A. phagocytophilum persistance. Nevertheless, no observation suggests that A. phagocytophilum could induce Direct Initial Interactions with Endothelial Cells any anti-apoptotic effect in ECs. ECs infections have been studied in vivo for A. marginale, using splenectomised calves [195], adult cows CONCLUSION AND FUTURE RESEARCH DIRECTIONS [196] or experimentally infected SCID mice [197]. Different techniques have been used (mainly co-localization with ECs Because ECs line the vasculature, their position allows markers) but none of them yielded unequivocal evidence of them to interact with any pathogen that can access the A. marginale infection of ECs in vivo [196]. circulating blood and its cells. A few of them, namely In the case of A. phagocytophilum, Munderloh et al. Anaplasma, Bartonella, Orientia and Rickettsia, are able to interact intimately with ECs of the micro-vascularization, [193] have shown that bovine and primate microvascular  endothelial cell lines are rapidly infected by this bacterium, and all belong to -Proteobacteria, are vector-borne and are which replicates intracellularly at high levels, whereas other relatively close. cell lines, including macrovascular aortic ECs, did not Limited data are available for Anaplasma, Orientia or support continuous development of this bacterium. Rickettsia concerning their relationships with ECs. On the Moreover, Herron et al. [197] have studied the initial contrary, studies on Bartonella-ECs interactions are infection of ECs by A. phagocytophilum in a mouse model numerous, due to the unique and remarkable ability of some after tick bite combined with in vitro assays. They also species of Bartonella to induce angiogenesis. Interestingly, observed a rapid colonisation of HMEC-1 (human the genus Bartonella is the only genus among all these microvascular ECs-1). groups that does not belong to the order Rickettsiales and that is not strictly intra-cellular. Secondary Interactions with Endothelial Cells After PMN Adhesion When all these bacterial groups are considered, the diversity of their effects on ECs, which are clearly target Choi et al. [198], tested the interactions of A. cells for these bacteria, is striking: all degrees of phagocytophilum alone with PMN and ECs. In their consequences are observed (Fig. 1). In all cases, their infection model, A. phagocytophilum-infected neutrophils interactions with ECs contribute, directly or indirectly, to the demonstrated a reduced capacity for binding to ECs. This vascular lesions observed during the course of the respective reduced capacity was closely linked to L-selectin and diseases. In most cases, the severity of the lesions is extracellular P-selectin glycoprotein ligand 1 (PSGL-1, correlated to the immunological status of the patient. But the CD162) domains shedding. According to these authors, the ability to exert an effect on ECs apoptosis and/or anti- adhesion defect associated with this shedding could lead to a apoptosis appears limited to some bacteria belonging to the prolonged functional incapacity of the infected PMN and Rickettsiaceae and Bartonellaceae family, and some thus to increased numbers of infected cells and their members of the last one are the only bacteria ever described persistence in the blood prior to tick bites. Some months that have been found able to induce an angiogenic effect. later, Park et al. [6] showed that this inhibition of neutrophil transmigration through the micro-endothelial barrier is If some arguments (i.e. persistent infection, long lasting observed with A. phagocytophilum-infected PMN but not bacteremia) are in favour of the hypothesis that ECs could with E. chaffeensis, which conversely increases the represent reservoir cells for these four genera, there is no transmigration of infected monocytes. These authors definitive proof of their persistance and long term replication conclude that this unique interaction of PMN and ECs is in the ECs of chronically infected animals. Moreover, the probably very important for this pathogen survival, cells used and/or the conditions of culture do not allow pathogenesis, and disease induction. considering the results obtained in vitro as fully reliable, as most studies have privileged HUVEC, which do not appear These results seem somewhat in contradiction with those as relevant as target ECs for arthropod borne bacteria belong obtained by Grab et al. [199] and Nayrko et al. [200], in a to the micro-vascularization. When ECs of the micro- co-infection model. According to them, A. phagocytophilum- vascularization have been included in in vitro studies, the infected neutrophils augment the trans-endothelial cell physiological conditions do not correspond to those that are migration of B. burgdorferi. The compromised integrity met, in vivo for ECs of the micro-vascularization, such as could be due to the actions of matrix metalloproteases hypoxia, temperature, gaz composition, dynamic shear stress (MMPs) on tight junction proteins, but also to the production fluid flow… by infected neutrophils of chemokines (IL-8) and cytokines (IL-6), biologically active compounds with multiple effects, The case of Bartonella transcends the bacterial world to including enhanced changes in vascular permeability related join the exciting field of cell angiogenesis induction. For to alterations in the ECs cytoskeleton. these reasons, Bartonella spp. is one of the arthropod-borne bacteria for which knowledge on its interactions with ECs The results obtained by Herron et al. [197], who infected have made substantial and rapid progress during the past cells with A. phagocytophilum alone indicate that A. years. Despite the fact that the endothelium is a major target phagocytophilum rapidly transferred from infected HMEC-1 for the Bartonella-induced pathogenic response, few data human microvascular ECs to PMN, as over 50% of these have been reported about the cell surface receptors involved cells became infected within two hours of coincubation with in Bartonella adhesion and invasion. These bacterial HMEC-1. In their assay, PMN adhered to, polarized, and receptors could be involved not only in invasion but also in migrated upon infected endothelial monolayers, which drives the mitogenic and angiogenic stimulation of infected cells. Vascular Endothelium and Vector Borne Pathogen Interactions Current Immunology Reviews, 2012, Vol. 8, No. 3 243

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Received: June 6, 2010 Revised: February 10, 2011 Accepted: May 15, 2011