Fractalkine functions as a chemoattractant for osteoarthritis synovial fibroblasts and stimulates phosphorylation of mitogen-activated protein kinases and Akt.

Fractalkine (FKN/CX3CL1) has been detected in synovial fluids from osteoarthritis (OA) patients. Additionally, low-level expression of the FKN receptor, CX3CR1, has been demonstrated in OA synovial lining. This study aimed to determine a biological function for this ligand/receptor pair in OA and to assess a potential signalling mechanism for FKN in this predominant synovial lining cell type, using chemotaxis assays, Western blotting and F-actin staining. Chemotaxis assays demonstrate that the chemokine domain of FKN effectively induces migration of OA fibroblasts. Consistent with this finding, visualization of F-actin demonstrates that 1 or 10 nM FKN induces noticeable reorganization of cytoskeletal structure in OA fibroblasts after 30 min stimulation with a maximal enhancement at approximately 2 h. In addition, Western blotting analysis demonstrates that FKN stimulates phosphorylation of the mitogen-activated protein (MAP) kinases p38, c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK) 1/2 as well as the serine-threonine kinase Akt at Ser 473 and Thr 308. All these phosphorylation events occur in a time-dependent manner, with little or no activation within 1 min, and maximal activation occurring typically between 5 and 30 min. Moreover, inhibition of ERK 1/2 significantly reduces FKN-induced OA fibroblast migration. These results suggest that FKN is a novel chemoattractant for OA fibroblasts, consistent with FKN-induced alterations in cytoskeletal structure. In addition, FKN induces OA fibroblast signalling via the MAP kinases p38, JNK and ERK 1/2, as well as Akt.

[Comparative evaluation of the efficiency of the effect of very high frequency electromagnetic waves on platelet functional activity]. / Sravnitel'naia otsenka éffektivnosti vliianiia na funktsional'nuiu aktivnost' trombotsitov élektromagnitnykh KVCh-kolebanii.

A comparative analysis was made of the effect of two kinds of EMI MMD-radiation: EMI MMD-waves, generated by a vehicle "Jav-1 M" (42.2 and 53.5 HHz), and EMI MMD-waves exerting influence with frequencies of molecular spectrum of radiation and nitric oxide absorption (150.176-150.644 HHz), obtained with a specially created generator, with respect to their influence on the functional ability of platelets of unstable angina pectoris patients. It was shown that in vitro EMI MMD-fluctuations with frequencies of molecular spectrum of radiation and nitric oxide absorption exert a stronger inhibiting influence on the functional activity of platelets of unstable angina pectoris patients. Features of the action of various kinds of EMI MMD-effect on the activative-high-speed characteristics of platelet aggregation are shown.

[The effect of electromagnetic waves of very high frequency of molecular spectra of radiation and absorption of nitric oxide on the functional activity of platelets]. / Vliianie élektromagnitnykh KVCh-kolebanii na chastotakh molekuliarnykh spektrov izlucheniia i poglashcheniia okisi azota na funktsional'nuiu aktivnost' trombotsitov.

A study was made of the effect of electromagnetic EMI MMD-fluctuation on the frequencies of molecular spectra of radiation, and nitric oxide absorption under in vitro conditions on the functional activity of platelets in patients with unstable angina pectoris, with the help of a specially created generator. At amplitude-modulated and continuous modes of EMI MMD-irradiation of platelet-rich plasma for 5, 15 and 30 min the platelet functional activity decreases, which was shown up in reduction of their activation and fall of aggregative ability. The degree, to which platelet functional activity was inhibited, depended on the mode of irradiation and on duration of EMI MMD effect. The most obvious changes in platelet activation and in their readiness to aggregative response were observed at a continuous mode of irradiation within a 15 min interval.

Fractalkine: a novel angiogenic chemokine in rheumatoid arthritis.

Angiogenesis is an important aspect of the vasculoproliferation found in the rheumatoid arthritic (RA) pannus. We have previously implicated members of the CXC chemokine family as potent angiogenic mediators in RA. We investigated the possibility that the sole member of the CX(3)C chemokine family, fractalkine (fkn), induces angiogenesis and that fkn might mediate angiogenesis in RA. Recombinant human fkn significantly induced migration of human dermal microvascular endothelial cells (HMVECs), a facet of the angiogenic response, in the pmol/L range in a concentration-dependent manner (P < 0.05). Fkn also induced the formation of significantly more endothelial tubes on Matrigel than did a negative control (P < 0.05). Fkn significantly induced 2.3-fold more blood vessel growth than control in the in vivo Matrigel plug assays (P < 0.05). We identified HMVEC expression of the fkn receptor, CX(3)CR1. Next, we determined if RA synovial fluid (SF)-induced angiogenesis was fkn-dependent. SFs from six RA patients immunodepleted of soluble fkn induced 56% less migration of HMVECs than did sham-depleted RA SFs (P < 0.05). In vivo, immunodepletion of fkn from six RA SFs significantly inhibited their angiogenic activity in Matrigel plug assays (P < 0.05). Immunodepletion of fkn from five RA synovial tissue homogenates inhibited their ability to induce angiogenesis in in vivo Matrigel plug assays (P < 0.05). These results establish a new function for fkn as an angiogenic mediator and suggest that it may mediate angiogenesis in RA.

Fractalkine, a novel chemokine in rheumatoid arthritis and in rat adjuvant-induced arthritis.

OBJECTIVE: To examine the expression of the novel CX3C chemokine fractalkine (Fkn) and its receptor (CX3CR1) in rheumatoid arthritis (RA) and rat adjuvant-induced arthritis (AIA), a model of RA. METHODS: Immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and chemotaxis assays were used. RESULTS: In rat AIA, synovial tissue (ST) macrophages, fibroblasts, endothelial cells, and dendritic cells were Fkn immunopositive, whereas lymphocytes did not significantly express Fkn. Significant staining for CX3CR1 was found in ST macrophages, fibroblasts, and dendritic cells, whereas only a small percentage of endothelial cells stained for CX3CR1 in rat AIA. We immunolocalized Fkn to RA ST macrophages, fibroblasts, endothelial cells, and dendritic cells. We also found intense ST macrophage and dendritic cell staining for CX3CR1 in RA ST. Flow cytometry analysis of RA synovial fluid (SF) and peripheral blood revealed a greater percentage of monocytes expressing Fkn and CX3CR1 compared with T cells. By ELISA, we found significantly elevated soluble Fkn (sFkn) levels in RA SF compared with SF from patients with osteoarthritis or other forms of arthritis. By RT-PCR, we found enhanced expression of Fkn and CX3CR1 mRNA on day 18 in rat AIA, a time of pronounced inflammation in the rat joint. Soluble Fkn-depleted RA SF showed significantly decreased chemotactic activity for monocytes compared with sham-depleted RA SF. CONCLUSION: These results indicate that Fkn and its receptor are both expressed in RA and in rat AIA, and that sFkn is up-regulated in RA SF. Furthermore, our data suggest a new role for Fkn in monocyte chemotaxis in the inflamed RA joint.

Regulation of angiogenesis by the C-X-C chemokines interleukin-8 and epithelial neutrophil activating peptide 78 in the rheumatoid joint.

OBJECTIVE: Angiogenesis, the growth of new blood vessels, is vital to the ingress of inflammatory leukocytes in rheumatoid arthritis (RA) synovial tissue and to the growth and proliferation of RA pannus. The factors that mediate the growth of new blood vessels have not been completely defined. This study examined the ability of Glu-Leu-Arg (ELR)-containing chemokines to induce angiogenesis in the RA joint. METHODS: To reflect angiogenic activity in vivo, we selected a model using whole human synovial tissue rather than isolated cells. Tissues were examined by immunohistochemistry and enzyme-linked immunosorbent assay, and tissue homogenates were immunoneutralized and assayed for their ability to induce endothelial cell chemotaxis and rat corneal neovascularization. RESULTS: Cells expressing interleukin-8 (IL-8) and epithelial neutrophil activating peptide 78 (ENA-78) were located in proximity to factor VIII-related antigen-immunopositive endothelial cells. RA homogenates produced more IL-8 and ENA-78 compared with normal synovial tissue homogenates. Moreover, homogenates from RA synovial tissue produced significantly more chemotactic activity for endothelial cells in vitro and angiogenic activity in the rat cornea in vivo than did normal synovial tissue homogenates. The effects of IL-8 and ENA-78 accounted for a significant proportion of the chemotactic activity of endothelial cells and angiogenic activity found in RA synovial tissue homogenates. CONCLUSION: These results indicate that the ELR-containing chemokines IL-8 and ENA-78 are important contributors to the angiogenic activity found in the inflamed RA joint. It is possible that efforts aimed at down-regulating these chemokines offer a novel targeted therapy for the treatment of RA.

IL-4 adenoviral gene therapy reduces inflammation, proinflammatory cytokines, vascularization, and bony destruction in rat adjuvant-induced arthritis.

IL-4 is a cytokine with anti-inflammatory properties on activated macrophages. Rheumatoid arthritis, an autoimmune inflammatory disease, is characterized by a paucity of IL-4 and an abundance of synovial macrophage-derived mediators. Herein, the effect of a single injection of adenovirus-producing rat IL-4 (AxCAIL-4) or a control virus with no inserted gene was compared with the effect of PBS injection into rat ankles. Ankles were injected before arthritis onset or at maximal inflammation. Preventatively, AxCAIL-4 reduced adjuvant-induced arthritis (AIA)- and/or AIA/adenoviral-induced ankle inflammation, decreasing articular index scores, ankle circumferences, paw volumes, radiographic scores, mean levels of monocyte chemoattractant protein-1, the number of inflammatory cells, and the number of synovial blood vessels. Therapeutically, AxCAIL-4 also decreased ankle circumferences and paw volumes in comparison with a control virus with no inserted gene and PBS groups. After arthritis onset, mean levels of TNF-alpha, IL-1beta, macrophage inflammatory protein-2, and RANTES were decreased in AxCAIL-4 rat ankle homogenates compared with PBS-treated homogenates. Thus, increased expression of IL-4 via gene therapy administered in a preventative and/or therapeutic manner reduced joint inflammation, synovial cellularity, levels of proinflammatory cytokines, vascularization, and bony destruction in rat AIA, suggesting that a similar treatment in humans may be beneficial.

Regulation of apoptosis and cell cycle activity in rheumatoid arthritis.

The regulation of proliferation and cell death is vital for homeostasis, but the mechanisms that coordinately balances these two events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining increases through enhanced proliferation, migration, and/or decreased cell death. The aberrant decrease in apoptosis or increased cell cycle activity of fibroblast-like or macrophage-like synoviocytes is responsible for the synovial hyperplasia and contributes to the destruction of cartilage and bone. Recently, numerous molecules that modulate apoptosis and cell cycle have been implicated to play a role in RA. This review will describe the current understanding of the molecular mechanisms that govern apoptosis and cell cycle and their relationship to RA pathogenesis.

[Information EHF-interactions in a system of live objects (human platelets)]. / Informatsionnoe KVCh-vzaimodeistvie v sisteme zhivykh ob''ektov (trombotsitov cheloveka).

The molecular informational interaction has been first detected in a system that involves human platelets, exposed to electromagnetic EHF-fluctuations at frequencies of molecular spectra of radiation and absorption of nitric oxide (150.176-150.644 HHz), and native platelets. It has been established that the incubation of a native platelet rich plasma with a similar plasma, exposed to a 5-minute effect of electromagnetic EHF-fluctuations at frequencies of molecular spectra of radiation and absorption of nitric oxide at a mode of peak and frequent modulation of a signal under in vitro conditions, causes a significant (P < 0.05) inhibition of platelet functional activity in the native plasma, in comparison with control. This was displayed by a decreased platelet activation and falling platelet aggregation ability. Some possible mechanisms of interaction are suggested to explain the described effect.

Temporal expression of inflammatory cytokines and chemokines in rat adjuvant-induced arthritis.

OBJECTIVE: To examine cytokine and chemokine production during the evolution of rat adjuvant-induced arthritis (AIA), a model of rheumatoid arthritis. METHODS: Clinical and laboratory assessment of the course of AIA was performed over a 47-day period. Levels of the cytokines tumor necrosis factor a (TNFalpha), interleukin-1beta (IL-1beta), and IL-6, as well as levels of the chemokines macrophage inflammatory protein 1alpha (MIP-1alpha) and JE, the murine homolog of monocyte chemoattractant protein 1, were determined by enzyme-linked immunosorbent assay in the sera and joints of AIA and control rats. Synovia from AIA rats were (immuno)histochemically analyzed. Results of cytokine and chemokine measurements were correlated with clinical and laboratory markers of inflammation and histology. RESULTS: Early (before day 14 post adjuvant injection) and later phases of AIA could be distinguished. Cytokine and chemokine production was increased in AIA versus control rats. The production of TNFalpha, IL-1beta, MIP-1alpha, and, as determined earlier, epithelial neutrophil-activating peptide 78-like protein was abundant prior to and during the course of AIA, while that of IL-6 and JE was elevated in the late phase of AIA. Cytokine and chemokine levels were correlated with the clinical symptoms of arthritis and blood neutrophil counts. Joint levels of IL-1beta showed correlation with synovial lining proliferation and neutrophil ingress into AIA synovium. CONCLUSION: Cytokines and chemokines are involved in the clinical, laboratory, and histologic changes underlying AIA. The production of these mediators may be temporally and spatially regulated. These findings may be important for the optimal timing of cytokine and chemokine targeting.

Soluble vascular cell adhesion molecule 1 mediation of monocyte chemotaxis in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by infiltration of leukocytes, including monocyte/ macrophages, into synovial tissue (ST), but factors mediating the ingress of these cells are poorly understood. Vascular cell adhesion molecule 1 (VCAM-1) plays an important role in adhesion of leukocytes to the vasculature. This study was undertaken to test the hypothesis that soluble VCAM-1 (sVCAM-1) might mediate chemotaxis of monocytes in RA. METHODS: Chemotaxis assays were performed using a modified Boyden chamber to determine the effects of sVCAM-1 on and the role of very late activation antigen 4 (VLA-4) in peripheral blood (PB) monocyte migration. Synovial fluids (SF) were immunodepleted of sVCAM-1 to identify a role for sVCAM-1 in RA. Immunohistochemistry and flow cytometry analyses were performed to show the expression of VLA-4 in ST, SF, and PB. Tyrosine phosphorylation was studied by Western blot analysis on PB monocyte lysates in the presence of signaling inhibitors. RESULTS: Soluble VCAM-1 induced monocyte migration in the nM range, in a concentration-dependent manner. Anti-VLA-4 significantly inhibited sVCAM-1-induced monocyte migration, suggesting that sVCAM-1 acts in part via a VLA-4-dependent mechanism. In RA SF, incubation with anti-VCAM-1 resulted in a reduction in the ability to induce monocyte migration (mean 28%). VLA-4 immunolocalized to RA ST, SF, or PB, monocytes, macrophages, and lymphocytes. Soluble VCAM-1 stimulated tyrosine phosphorylation in monocytes, and pertussis toxin, chelerythrine chloride, and staurosporine significantly reduced sVCAM-1-mediated monocyte chemotaxis, suggesting that signaling pathways via G proteins and protein kinase C are required for sVCAM-1-mediated monocyte migration. CONCLUSION: These results demonstrate a novel function for sVCAM-1 as a monocyte chemotactic agent in RA and suggest a new potential target for modulating monocyte ingress into inflamed RA ST.

Ley/H: an endothelial-selective, cytokine-inducible, angiogenic mediator.

Endothelial cells (ECs) are key participants in angiogenic processes that characterize tumor growth, wound repair, and inflammatory diseases, such as human rheumatoid arthritis (RA). We and others have shown that EC molecules, such as soluble E-selectin, mediate angiogenesis. Here we describe an EC molecule, Lewisy-6/H-5-2 glycoconjugate (Ley/H), that shares some structural features with the soluble E-selectin ligand, sialyl Lewisx (sialyl Lex). One of the main previously recognized functions of Lewisy is as a blood group glycoconjugate. Here we show that Ley/H is rapidly cytokine inducible, up-regulated in RA synovial tissue, where it is cell-bound, and up-regulated in the soluble form in angiogenic RA compared with nonangiogenic osteoarthritic joint fluid. Soluble Ley/H also has a novel function, for it is a potent angiogenic mediator in both in vitro and in vivo bioassays. These results suggest a novel paradigm of soluble blood group Ags as mediators of angiogenic responses and suggest new targets for therapy of diseases, such as RA, that are characterized by persistent neovascularization.

Treatment with sulfasalazine or sulfapyridine, but not 5-aminosalicyclic acid, inhibits basic fibroblast growth factor-induced endothelial cell chemotaxis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by leukocyte recruitment and angiogenesis. We investigated the effects of sulfasalazine (SSZ) and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), on components of angiogenesis, namely, endothelial cell (EC) chemotaxis and proliferation, as well as on EC chemokine and soluble adhesion molecule expression. METHODS: SSZ, SP, and 5-ASA were assayed for their effects on basic fibroblast growth factor (bFGF)-induced human dermal microvascular endothelial cell (HMVEC) chemotaxis and proliferation. EC were plated on Matrigel to assess the effect of SSZ on EC tube formation. Enzyme-linked immunosorbent assays were performed to determine changes in HMVEC production of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), growth-related oncogene alpha (GROalpha), epithelial neutrophil-activating peptide 78 (ENA-78), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule 1 (sICAM-1) upon treatment with SSZ or its metabolites. RESULTS: HMVEC incubated with SSZ or SP exhibited reduced bFGF-induced chemotaxis (59%, [n = 7] and 22%, [n = 3], respectively) (P<0.05). SSZ and SP decreased basal HMVEC proliferation, while 5-ASA increased proliferation (P<0.05; [n = 5]). SSZ decreased bFGF-induced HMVEC proliferation (P<0.05 [n = 5]). SSZ inhibited phorbol 12-myristate 13-acetate-induced HMVEC tube formation (P<0.05; [minimum n = 5]). Tumor necrosis factor alpha-stimulated HMVEC shedding of sICAM-1 was reduced by incubation with either SSZ (19%) or 5-ASA (23%) (P<0.05; [n = 6]). SP inhibited cytokine-stimulated HMVEC expression of IL-8 and MCP-1 (P<0.05; [n = 4]). Neither SSZ nor its metabolites had any effect on HMVEC production of sE-selectin, GROalpha, or ENA-78. CONCLUSION: These results demonstrate that SSZ and its metabolite SP may affect the pathogenesis of RA by inhibiting EC chemotaxis, proliferation, tube formation, and expression of sICAM-1, IL-8, and MCP-1.

PECAM-1 and leukosialin (CD43) expression correlate with heightened inflammation in rat adjuvant-induced arthritis.

A hallmark of both adjuvant-induced arthritis (AIA) and rheumatoid arthritis is chronic joint inflammation characterized by ingress of leukocytes into the inflamed synovial tissue. The timing of expression of adhesion molecules, which govern the ingress of leukocytes, is important in the orchestration of an inflammatory response. We examined the expression of vascular cell adhesion molecule-1 (VCAM-1), sialo adhesin, platelet and endothelial cell adhesion molecule-1 (PECAM-1), and leukosialin (CD43) in AIA, starting at adjuvant injection (day 0), through the peak of inflammation (day 18 postadjuvant injection), until day 54. VCAM-1 is constitutively expressed on the lining layer and ECs and its expression levels do not change throughout the progression of AIA. Sialoadhesin synovial tissue lining cell expression is decreased after adjuvant injection. In contrast, PECAM-1 expression is increased on synovial tissue lining cells on day 7 and is elevated through day 54 (peaking on day 54 with six-fold more cells expressing PECAM-1). PECAM-1 expression on endothelial cells peaks on day 7 with three-fold more cells expressing it, while on macrophages expression maximizes on day 25 with six-fold more cells expressing PECAM-1. CD43 expression is increased on synovial tissue lining cells, macrophages, neutrophils, and lymphocytes on days 18 and 25, before going back to basal levels. The increased expression of PECAM-1 and CD43 on leukocytes at the height of inflammation in AIA suggests important roles for these adhesion molecules in potentially binding their EC ligands resulting in leukocyte ingress into the synovial tissue.

The role of an epithelial neutrophil-activating peptide-78-like protein in rat adjuvant-induced arthritis.

The chemokine, epithelial neutrophil-activating peptide-78 (ENA-78), is a potent neutrophil chemotaxin whose expression is increased in inflamed synovial tissue and fluid in human rheumatoid arthritis compared with osteoarthritis. Since ENA-78 has been implicated in the pathogenesis of RA, we examined the expression of an ENA-78-like protein during the development of rat adjuvant-induced arthritis (AIA). Using an ELISA assay, we found increased levels of antigenic ENA-78-like protein in the sera of AIA animals compared with control normal animals by day 7 postadjuvant injection. ENA-78-like protein levels continued to increase as AIA developed. ENA-78-like protein levels in joint homogenates were increased in AIA animals later in the development of the disease, by day 18 during maximal arthritis, compared with control animals. Expression of ENA-78-like protein in both the AIA serum and joint correlated with the progression of inflammation of the joints. Anti-human ENA-78 administered before disease onset modified the severity of AIA, while administration of anti-ENA-78 after clinical onset of AIA did not modify the disease. These data support a role for an ENA-78-like protein as an important chemokine in the progression and maintenance of AIA.

RANTES expression and contribution to monocyte chemotaxis in arthritis.

Rheumatoid arthritis (RA) is characterized by recruitment of leukocytes from the vasculature into inflamed synovial tissue (ST) and synovial fluid (SF), which depends, in part, upon the continued maintenance of chemotactic stimuli. RANTES is a potent chemoattractant for leukocytes including monocytes and CD45RO+ memory T lymphocytes. The aim of this study was to determine the production, the source, and the function of antigenic RANTES in arthritis. We detected antigenic RANTES in SFs from RA and OA patients (100 +/- 22.7 and 72 +/- 30.7 pg/ml, respectively). CM from RA ST fibroblasts stimulated with interleukin-1beta or tumor necrosis factor-alpha contained significantly more antigenic RANTES than unstimulated CM (452 +/- 181.6 and 581 +/- 200.2 pg/ml, respectively, versus 12 +/- 4.4 pg/ml, P < 0.05). PHA-stimulated RA SF mononuclear cells secreted 5- to 15-fold more antigenic RANTES than did nonstimulated mononuclear cells, while LPS induced secretion up to 4-fold. We immunolocalized antigenic RANTES to sublining macrophages (28 +/- 3.7 and 8 +/- 2.0% immunopositive cells), perivascular macrophages (56 +/- 6.9 and 19 +/- 3.4%), and synovial lining cells (37 +/- 5.8 and 60 +/- 10.4%) in RA and OA tissue, respectively. Anti-RANTES neutralized 20.2 +/- 1.3% of the RA SF chemotactic activity for normal peripheral blood monocytes (P < 0.05). These results demonstrate antigenic RANTES in RA and OA ST and SF and identify RANTES as a chemoattractant for monocytes in the RA joint.

Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing.

Angiogenesis is an essential component of normal wound repair, yet the primary mediators of wound angiogenesis have not been well described. The current study characterizes the contribution of vascular endothelial cell growth factor (VEGF) to the angiogenic environment of human surgical wounds. Surgical wound fluid samples (n = 70) were collected daily for up to 7 postoperative days (POD) from 14 patients undergoing mastectomy or neck dissection. VEGF levels in surgical wound fluid were lowest on POD 0, approximating values of serum, but increased steadily through POD 7. An opposite pattern was noted for basic fibroblast growth factor-2. Fibroblast growth factor-2, which has been previously described as a wound angiogenic factor, exhibited highest levels at POD 0, declining to near serum levels by POD 3. Surgical wound fluid form all time points stimulated marked endothelial cell chemotaxis and induced a brisk neovascular response in the rat corneal micropocket angiogenesis assay. Antibody neutralization of VEGF did not affect the in vitro chemotactic or the in vivo angiogenic activity early wound samples (POD 0). In contrast, VEGF neutralization significantly attenuated both chemotactic activity (mean decrease 76 +/- 13%, P < 0.01) and angiogenic activity (5 of 5 samples affected) of later wound samples (POD 3 and 6). The results suggest a model of wound angiogenesis in which an initial angiogenic stimulus is supplied by fibroblast growth factor-2, followed by a subsequent and more prolonged angiogenic stimulus mediated by VEGF.

Chemokine receptor CXCR4 expression in endothelium.

The expression of chemokine receptor and viral coreceptor CXCR4 is reported in cultured endothelial cells and in arterial endothelium. A 1.9 kb transcript was cloned from cultured bovine aortic (BAEC) and human umbilical vein endothelial cells (HUVEC). CXCR4 mRNA was expressed at high levels in BAEC and HUVEC but was not expressed by cultured bovine arterial smooth muscle cells (BASM) or human umbilical vein smooth muscle cells (HUVSM). Western blotting with polyclonal antibodies demonstrated an approximate 46KD protein in endothelial cells only. In situ hybridization and immunocytochemistry (anti-CXCR4 monoclonal antibody 12G5) revealed both transcript and protein expression in cultured endothelial cells, and in the endothelium of normal aorta but not in aortic smooth muscle. The ligand for CXCR4, stromal cell derived factor 1 (SDF-1) stimulated mobilization of intracellular calcium at a moderate level (37% of the peak response to thrombin), confirming the expression of functional receptor at the endothelial surface. The involvement of CXCR4 in chemokine signaling, chemoattraction (through SDF-1), and its potential viral coreceptor activity suggest a multifunctional role in vascular homeostasis and pathophysiology.

Spatial relationships in early signaling events of flow-mediated endothelial mechanotransduction.

Blood flow interactions with the vascular endothelium represent a specialized example of mechanical regulation of cell function that has important physiological and pathological cardiovascular consequences. The endothelial monolayer in vivo acts as a signal transduction interface for forces associated with flowing blood (hemodynamic forces) in the acute regulation of artery tone and chronic structural remodeling of arteries, including the pathology of atherosclerosis. Mechanisms related to spatial relationships at the cell surfaces and throughout the cell that influence flow-mediated endothelial mechanotransduction are discussed. In particular, flow-mediated ion channel activation and cytoskeletal dynamics are considered in relation to topographic analyses of the luminal and abluminal surfaces of living endothelial cells.

Gap junctional communication between vascular cells. Induction of connexin43 messenger RNA in macrophage foam cells of atherosclerotic lesions.

The structure and function of blood vessels depend on the ability of vascular cells to receive and transduce signals and to communicate with each other. One means by which vascular cells have been shown to communicate is via gap junctions, specifically connexin43. In atherosclerosis, the normal physical patterns of communication are disrupted by the subendothelial infiltration and accumulation of blood monocytes, which in turn can differentiate into resident foam cells. In this paper we report that neither freshly isolated human peripheral blood monocytes nor differentiated monocytes/macrophages exhibit functional gap junctional dye transfer in homo-cellular culture or in co-culture with endothelial cells or smooth muscle cells. By Northern analysis, neither freshly isolated blood monocytes nor pure cultures of differentiated monocyte/macrophages expressed gap junction messenger RNA. However, immunohistochemical staining followed by in situ hybridization on sections of human atherosclerotic carotid arteries revealed strong expression of gap junction connexin43 messenger RNA by macrophage foam cells. These results suggest that tissue-specific conditions present in atherosclerotic arteries induce expression of connexin43 messenger RNA in monocyte/macrophages.