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PubMed2004

Immunohistochemical localization of alphavbeta3 integrin receptor during experimental tooth movement.

During orthodontic treatment, multinucleated clast cells carry out the resorption of mineralized tissues. Adhesion of clast cells to the mineralized tissues is mediated by transmembrane cell-surface glycoproteins called integrins, specifically by the alphavbeta3 integrin, which plays an important role in the process of bone resorption. The role of the alphavbeta3 integrin in bone resorption leading to osteoporosis has been demonstrated, but its role in alveolar bone and root resorption during orthodontic tooth movement is unknown. This study examined the expression of the alphavbeta3 integrin during experimental tooth movement. Tooth movement was achieved in 16 male Sprague-Dawley rats (each weighing 120-200 g) with elastic bands between their maxillary first and second molars. The molar-bearing segments were dissected and processed for histologic and immunohistochemical examination. The expression of alphavbeta3 integrin was examined with 2 primary antibodies: a polyclonal anti-alphav integrin subunit antibody and a polyclonal anti-beta3 integrin subunit antibody. Negative controls were similarly processed but without incubation with primary antibodies. The alphavbeta3 integrin was expressed both by osteoclasts associated with alveolar bone resorption and by odontoclasts associated with root resorption during experimental tooth movement. Furthermore, the beta3 integrin subunit was expressed by the epithelial rests of Malassez in the periodontal ligament. Negative controls did not show immunolabeling. The alphavbeta3 integrin adhesion receptor is expressed during experimental tooth movement and might be involved in the process of mineralized tissue resorption and the functions of the epithelial rests of Malassez.

Alveolar Process↗
PubMed1998

Mitotic inhibition of corneal endothelium in neonatal rats.

PURPOSE: Corneal endothelium in humans does not divide to any significant extent after birth; therefore, with age there is a gradual loss of cells. When cell density is reduced to a critical level, the endothelium cannot function to maintain corneal clarity, and the cornea becomes permanently cloudy. Currently, the blindness that results can be treated only by corneal transplantation. The long-term goal is to find methods to stimulate corneal endothelial proliferation in a clinically relevant manner. The first step toward achieving this goal is to identify mechanisms responsible for the induction and maintenance of mitotic inhibition of the corneal endothelium in vivo. During corneal development, the endothelium is formed by migration and proliferation of mesenchymal cells from the ocular periphery. Soon after the monolayer is formed, proliferation ceases. In tissue culture, many cell types cease proliferating upon formation of stable cell-cell and cell-substrate attachments. The goal of the present studies was to determine whether establishment of stable contacts correlates with cessation of endothelial proliferation during corneal development in vivo. METHODS: Corneas from neonatal (days 1, 3, 7, 10, 13, 14, 17, 21, 28, and 42) and adult rats were used for immunolocalization of the following: bromodeoxyuridine (BrdU), an S-phase marker; p27kip1 and p21cip1, G1-phase inhibitors; connexin-43 and ZO-1, proteins associated with gap and tight junctions, respectively; Na+/K+-ATPase and beta3-integrin, markers of plasma membrane polarity; and fibronectin and collagen type IV, constituents of Descemet's membrane. Nuclei staining positively for BrdU were counted to determine the relative number of S-phase cells at various times after birth. Marker protein expression and localization were determined by conventional fluorescence microscopy and by confocal microscopy. RESULTS: The number of endothelial cells staining positively for BrdU gradually decreased between postnatal days 1 and 13. After postnatal day 13, positive BrdU staining was no longer detectable. During the first postnatal week, cells stained positively for the G1-phase inhibitor p27kip1 but not for p21cip1. Connexin-43 achieved its mature location by postnatal day 1. ZO-1, Na+/K+-ATPase, beta3-integrin, fibronectin, and collagen type IV achieved their mature localization patterns between postnatal days 14 and 21. CONCLUSIONS: In neonatal rat, corneal endothelial cells are still entering the cell cycle at birth, but cell cycle entry gradually decreases, so that by postnatal day 13 cells are no longer entering the S-phase. The G1-phase inhibitor p27kip1, but not p21cip1, may help mediate this inhibition. Stable cell-cell and cell-substrate contacts gradually form, and monolayer maturation is complete between postnatal days 14 and 21. The results lead to the hypothesis that, in developing rat cornea in vivo, the establishment of stable cell-cell and cell-substrate contacts initiates a cascade of events, mediated by p27kip1, which induces mitotic inhibition in the endothelial monolayer.

Animals↗
PubMed2006

Integrin signaling is critical for pathological angiogenesis.

The process of postnatal angiogenesis plays a crucial role in pathogenesis of numerous diseases, including but not limited to tumor growth/metastasis, diabetic retinopathy, and in tissue remodeling upon injury. However, the molecular events underlying this complex process are not well understood and numerous issues remain controversial, including the regulatory function of integrin receptors. To analyze the role of integrin phosphorylation and signaling in angiogenesis, we generated knock-in mice that express a mutant beta3 integrin unable to undergo tyrosine phosphorylation. Two distinct models of pathological angiogenesis revealed that neovascularization is impaired in mutant beta3 knock-in mice. In an ex vivo angiogenesis assay, mutant beta3 knock-in endothelial cells did not form complete capillaries in response to vascular endothelial growth factor (VEGF) stimulation. At the cellular level, defective tyrosine phosphorylation in mutant beta3 knock-in cells resulted in impaired adhesion, spreading, and migration of endothelial cells. At the molecular level, VEGF stimulated complex formation between VEGF receptor-2 and beta3 integrin in wild-type but not in mutant beta3 knock-in endothelial cells. Moreover, phosphorylation of VEGF receptor-2 was significantly reduced in cells expressing mutant beta3 compared to wild type, leading to impaired integrin activation in these cells. These findings provide novel mechanistic insights into the role of integrin-VEGF axis in pathological angiogenesis.

Animals↗
PubMed2003

Leptin regulation of the interleukin-1 system in human endometrial cells.

We have previously shown that (i). leptin and leptin receptor (Ob-R) are expressed in the human endometrium, and (ii). leptin secretion is regulated in blastocyst and endometrial epithelial cell (EEC) co-cultures. Interleukin-1beta (IL-1beta) up-regulates leptin and Ob-R, and both cytokines up-regulate beta3 integrin expression in EEC. In the present investigation we examined the effect of leptin on the expression of the IL-1 system in EEC and endometrial stromal cells (ESC) cultured in a medium containing insulin, leptin or IL-1beta (0-3 nmol/l). Leptin stimulated IL-1 antagonist (IL-1Ra), IL-1beta secretion and expression of IL-1 receptor type I (IL-1R tI) in both cell types. IL-1beta and IL-1Ra secretion were down-regulated by IL-1R tI blockade using specific antibodies. Interestingly, leptin partially neutralized this effect. The blockade of Ob-R neutralized the effects of both leptin and IL-1beta on expression of the IL-1beta system and beta3 integrin and on phosphorylation of signal transducer and activator of transcription 3 (Stat3). These results suggest that leptin regulates the IL-1 system and that the blockade of functional Ob-R impairs leptin and IL-1beta functions at the endometrial level. Leptin could be an important molecule for implantation and a molecular mediator for actions of the IL-1 system. The fact that leptin, in the absence of IL-1, can trigger the expression of markers of endometrial receptivity and of the invasive trophoblast phenotype (as does IL-1), suggest that leptin could substitute for these IL-1 functions during the implantation process.

Antibodies↗
PubMed2004

A variant thrombasthenic phenotype associated with compound heterozygosity of integrin beta3-subunit: (Met124Val)beta3 alters the subunit dimerization rendering a decreased number of constitutive active alphaIIbbeta3 receptors.

We report the analysis of a variant case of thrombasthenic phenotype that is a compound heterozygote for two mutations located within the metal ion dependent adhesion site (MIDAS) of the beta3 subunit. The patient inherited a maternal allele carrying the Met124Val substitution and a paternal allele that changes Asp119 to Tyr. Phenotyping of the human platelet antigen 1 (HPA-1) showed that the platelet alphaIIbbeta3 complex in the patient was mostly accounted for by the Asp 119Tyr allele that does not bind to fibrinogen (Fg). The patient showed agonistinduced binding of platelets to Fg but neither binding to PAC-1 nor cell aggregation could be detected, most likely due to the minute expression (< or = 5%) of alphaIIb(124Val)beta3 receptors. CHO cells expressing (124Val)beta3 showed a diminished surface expression of alphaIIbbeta3, enhanced adhesion to immobilized Fg, and spontaneous aggregation in the presence of soluble Fg, suggesting that (124Val)beta3 may confer constitutive activity to the alphaIIb(124Val)beta3 receptors. A distinct feature of these cells is the failure of DTT to enhance the binding to soluble Fg and the formation of cell aggregates. The substitution of (124Met)beta3 by either a polar or a positively charged amino acid restored the surface exposure and function of the alphaIIbbeta3 receptors whereas a negatively charged residue did not.

Alleles↗
PubMed2004

Leptin serves as an upstream activator of an obligatory signaling cascade in the embryo-implantation process.

Leptin is essential for mouse reproduction, but the exact roles it serves are yet to be determined. Treatment of cultured endometrial cells with leptin increases the level of beta3-integrin, IL-1, leukemia inhibitory factor, and their corresponding receptors. These leptin-induced effects are eliminated by inhibitors of leptin receptor (OB-R) signaling. Herein the impact of blocking leptin/OB-R signaling in the mouse endometrium was assessed. Intrauterine injection of either leptin peptide antagonists (LPA-1 or -2) or OB-R antibody on d 3 of pregnancy impaired mouse implantation in comparison to intrauterine injection of scrambled peptides (LPA-Sc) or species-matched IgGs. Significant reduction in the number of implantation sites and uterine horns with implanted embryos was found after intrauterine injection of LPA-1 (1 of 22) vs. LPA-1Sc (11 of 15) and LPA-2 (3 of 17) vs. LPA-2Sc (14 of 16). The impact of disruption of leptin signaling on the endometrial expression of several molecules in pregnant mice was assessed by Western blot, immunohistochemistry, and confocal microscopy. Disruption of leptin signaling resulted in a significant reduction of IL-1 receptor type I, leukemia inhibitory factor, vascular endothelial growth factor receptor 2, and beta3-integrin levels. The levels of colony stimulating factor-1 receptor and OB-R were unaltered after treatment with LPAs compared with controls. Expression of OB-R protein was pregnancy dependent and found only in glandular epithelium after implantation occurred. Our findings support previous observations that leptin signaling is critical to the implantation process and suggest that molecules downstream of leptin-activated receptor may serve obligatory roles in endometrial receptivity and successful implantation.

Animals↗
PubMed1998

Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase.

Thrombospondin-1 is a matrix protein that inhibits proliferation, motility and sprouting of endothelial cells in vitro and angiogenesis in vivo. One mechanism by which thrombospondin-1 may influence endothelial cell biology is through interaction with the endothelial cell alphav beta3 integrin receptor. This interaction is mediated via a cryptic Arg-Gly-Asp sequence in the C-terminal Ca2+-binding region of thrombospondin-1. Exposure of the Arg-Gly-Asp sequence is controlled by disulfide interchange events in the Ca2+-binding loops and C-globular domain. Limited reduction of thrombospondin-1 by dithiothreitol exposes the Arg-Gly-Asp sequence which can bind to the alphav beta3 integrin receptor and support endothelial cell spreading (X. Sun, K. Skorstengaard, D.F. Mosher, J. Cell Biol. 118 (1992) 693-701). Our aim was to identify possible physiological reductants that can mediate Arg-Gly-Asp exposure. We now report that protein disulfide isomerase, which is known to catalyze disulfide interchange in thrombospondin-1 and change its enzyme inhibitory properties and its binding to monoclonal antibodies, was secreted by bovine aortic endothelial cells and deposited on the cell surface. There was an average of approximately 2.2 fg of protein disulfide isomerase on the surface of a bovine aortic endothelial cell. Treatment of thrombospondin-1 with purified protein disulfide isomerase enhanced adhesion of endothelial cells to thrombospondin-1 in an Arg-Gly-Asp-dependent manner through the alphav beta3 integrin receptor and supported cell spreading. Both Ca2+-depleted and Ca2+-replete thrombospondin-1 were substrates for protein disulfide isomerase. These results suggest that endothelial cell derived protein disulfide isomerase may regulate Arg-Gly-Asp-dependent binding of thrombospondin-1.

Animals↗
PubMed2002

Expression of osteopontin at sites of bone erosion in a murine experimental arthritis model of collagen-induced arthritis: possible involvement of osteopontin in bone destruction in arthritis.

OBJECTIVE: To investigate the involvement of osteopontin (OPN) in bone destruction in a murine experimental arthritis model of collagen-induced arthritis (CIA). METHODS: The expression of OPN was examined at both the messenger RNA (mRNA) and protein levels in various arthritic lesions in mice with CIA by in situ hybridization and immunohistochemistry, respectively. In addition, the expression of alpha(v)beta3 integrin, a receptor for OPN, the ligation of which is thought to be essential for bone resorption by osteoclasts, was examined by immunohistochemistry. Plasma concentrations of OPN were measured at different time points in the course of CIA by enzyme-linked immunosorbent assay. RESULTS: OPN mRNA was detected mainly at sites of bone erosion in arthritic lesions, where activated osteoclasts were present; OPN protein was also detected at sites of bone erosion. In the arthritic synovium, OPN was predominantly expressed in the synovial lining layer, but not in lymphoid aggregates. In addition, alpha(v)beta3 integrin was detected coincident with OPN at sites of bone erosion (bone-pannus junction). Plasma OPN levels were markedly elevated at the time points that corresponded to arthritis flares, and higher levels were maintained during the progression of arthritis. CONCLUSION: OPN may mediate bone resorption by osteoclasts in arthritis through ligation with its receptor, alpha(v)beta3 integrin. OPN may be a useful therapeutic target molecule in the prevention of bone destruction in arthritis.

Animals↗
PubMed2005

Variation in ITGB3 is associated with asthma and sensitization to mold allergen in four populations.

RATIONALE: Recent genetic studies have implicated integrins in asthma and atopy susceptibility. We therefore evaluated the integrin-beta3 gene (ITGB3), an integrin gene within an asthma linkage peak on chromosome 17, as a candidate for susceptibility to asthma- and atopy-related phenotypes. METHODS AND MEASUREMENTS: We genotyped and performed association tests on 19 single nucleotide polymorphisms in ITGB3 in the Hutterites, a founder population, and in three outbred replication populations. MAIN RESULTS: Variation in ITGB3 was strongly associated with susceptibility to bronchial hyperresponsiveness and protection from allergic sensitization to mold allergens in this population. Three independent case-control populations representing Caucasians and African Americans were used to replicate this finding, also revealing ITGB3 alleles that are associated with asthma susceptibility and protection from mold allergen sensitization. CONCLUSIONS: This study provides evidence that ITGB3 plays a role in the pathogenesis of asthma and sensitization to mold allergens.

Black or African American↗
PubMed2006

Homocysteine enhances bone resorption by stimulation of osteoclast formation and activity through increased intracellular ROS generation.

UNLABELLED: Hyperhomocystinemia is a modifiable risk factor for osteoporosis and fracture. Physiologic concentrations of Hcy directly activate osteoclast formation and activity through stimulation of p38 MAPK and integrin beta3. The effects of Hcy were mediated by generation of intracellular ROS. INTRODUCTION: Hyperhomocysteinemia is a modifiable risk factor for osteoporosis and its related bone fractures. It has been reported that bone resorption and turnover rate were increased in hyperhomocystinemia. Using mouse bone marrow cells, we examined the direct effects of homocysteine (Hcy) on osteoclast formation and activity. MATERIALS AND METHODS: Osteoclast formation was determined by TRACP staining and TRACP activity. Intracellular reactive oxygen species (ROS) generation was measured using a fluorescent probe, dichlorodihydrofluorescein diacetate. Intracellular signaling cascades of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-kappaB were measured by Western blotting. Integrin beta3 mRNA levels were measured by RT-PCR. Actin ring formation and bone resorption assays were also performed. RESULTS: Physiologic concentrations of Hcy upregulated TRACP+ multinucleated cells and TRACP activity, stimulated actin ring formation, and increased the number of nuclei per cell and the level of expression of integrin beta3 mRNA. In addition, Hcy increased bone resorption and stimulated p38 MAPK activity and intracellular reactive oxygen species (ROS) generation. All of these Hcy-induced changes were blocked by pretreatment with the antioxidant, N-acetyl cysteine. CONCLUSIONS: Hcy directly activates osteoclast formation and activity through increased generation of intracellular ROS. These findings suggest that, in individuals with mild to moderate hyperhomocystinemia, increased bone resorption by osteoclasts may contribute to osteoporosis and that an antioxidant may attenuate bone loss in these individuals.

Acid Phosphatase↗
PubMed2003

Vascular damage and lack of angiogenesis in systemic sclerosis skin.

The aim of this study was to analyse microvascular damage and compensatory angiogenesis in skin from patients with systemic sclerosis (SSc) compared with systemic lupus erythematosus (SLE), Raynaud's phenomenon (RP) and healthy controls. Immunohistochemistry was used for skin biopsies (9 SSc, 10 SLE, 9 RP and 12 healthy controls) using von Willebrand factor and beta3 integrin subunit specific antibodies, TechMate immunostaining robot and biotin-streptavidin protocol. In the early stages of SSc, vWF was found in the perivascular space and interstitial matrix in papillary but not in the reticular dermis, in particular around small oedematous blood vessels infiltrated by mononuclear cells. The extravascular release of vWF in SSc specimens was associated with weak or even a total lack of immunoreactivity within the associated endothelial cells. Late stages of SSc were characterised by loss of the dermal papillae, subepidermal fibrosis, hypovascularity and strong endothelial vWF expression without extravascular leakage. In all SSc patients studied only a few vascular profiles were weakly immunostained for beta3 integrin subunit. This work demonstrates that vWF is not only released into the systemic circulation, but is also leaked to the perivascular space/matrix. This local release and deposition of vWF is probably a sensitive and early marker of microvascular involvement in SSc pathogenesis. Local vWF release may play a role in platelet adhesion, aggregation, thrombogenesis and dermal connective tissue remodelling. In spite of some attempts towards compensatory angiogenesis in SSc, as evidenced by beta3 integrin subunit expression, it was evident that the angiogenic response was not able to prevent the development of hypovascularity during the advanced stages of the disease.

Biopsy, Needle↗
PubMed1998

The integrin ligand echistatin prevents bone loss in ovariectomized mice and rats.

Integrins that bind RGD (arginine-glycine-aspartic acid) containing peptides, especially the vitronectin receptor alpha(v)beta3, have been implicated in the regulation of osteoclast function. Echistatin, an RGD-containing snake venom peptide with high affinity for beta3 integrins, as well as nonpeptide RGD mimetics, were shown to inhibit osteoclastic bone resorption in vitro and in vivo. To evaluate the role of RGD-binding integrins in bone metabolism, we examined by several methods the effects of echistatin on ovariectomy (OVX)-induced bone loss in mice and rats. First, we confirmed that echistatin binds in vitro with high affinity (Kd, 0.5 nM) to alpha(v)beta3 integrin purified from human placenta and established a competitive binding assay to measure echistatin concentrations in serum. We find that echistatin infused for 2 or 4 weeks at 0.36 microg/h x g body weight (approximately 50 nmol/day x mouse) completely prevents OVX-induced cancellous bone loss in the distal femora of ovariectomized mice. Echistatin has no effect on uterine weight, body weight, and femoral length changes induced by OVX, nor does it cause any apparent changes in major organs other than bone. In OVX rats, echistatin infusion at 0.26 microg/h x g for 4 weeks effectively prevents bone loss, evaluated by dual energy x-ray absorptiometry of the femur, by femoral ash weight, and by bone histomorphometry of the proximal tibia. At effective serum concentrations of 20-30 nM, measured at the end of the infusion period, echistatin maintains histomorphometric indices of bone turnover at control levels but does not decrease osteoclast surface. In conclusion, these results provide in vivo evidence, at the level of bone histology, that RGD-binding integrins, probably alpha(v)beta3, play a rate-limiting role in osteoclastic bone resorption and suggest a therapeutic potential for integrin ligands in the suppression of bone loss.

Animals↗
PubMed2003

Regulatory mechanism of osteoclast activation.

Osteoclasts are multinucleated, terminally differentiated cells which play an essential role in bone resorption. Osteoclasts exhibit high expression of the alpha(v)beta3 integrin, which binds to a variety of extracellular matrix proteins, including vitronectin, osteopontin and bone sialoprotein. RGD (Aug-Gly-Asp)-containing peptides, RGD-mimetics and blocking antibodies to alpha(v)beta3 integrin were shown to inhibit bone resorption in vitro and in vivo, suggesting that this integrin plays an important role in regulating osteoclast function. A number of signalling molecules were found to be involved in the alpha(v)beta3 integrin-dependent signalling pathway, including c-Src, Pyk2 and p130Cas. Both Pyk2 and p130Cas localize to the sealing zone of actively resorbing osteoclasts, suggesting their role in linking the adhesion of osteoclasts to the bone matrix, to cytoskeletal organization, and to the polarization and activation of these cells for bone resorption. In this article, we review the regulatory mechanism of osteoclast activation.

Animals↗
PubMed1997

Truncation of the cytoplasmic domain of beta3 in a variant form of Glanzmann thrombasthenia abrogates signaling through the integrin alpha(IIb)beta3 complex.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by absence or dysfunction of the platelet integrin alpha(IIb)beta3. Patient RM is a thrombasthenic variant whose platelets fail to aggregate in response to physiological agonists, despite the fact that they express abundant levels of alpha(IIb)beta3 on their surface. Binding of soluble fibrinogen or fibrinogen mimetic antibodies to RM platelets did not occur, except in the presence of ligand-induced binding site (LIBS) antibodies that transformed the RM integrin complex into an active conformation from outside the cell. Sequence analysis of PCR-amplified genomic DNA and platelet mRNA revealed a C2268T nucleotide substitution in the gene encoding the integrin beta3 subunit that resulted in an Arg724Ter mutation, producing a truncated protein containing only the first eight of the 47 amino acids normally present in the cytoplasmic domain. Functional analysis of both RM platelets and CHO cells stably expressing this truncated integrin revealed that the alpha(IIb)beta3Arg724Ter complex is able to mediate binding to immobilized fibrinogen, though downstream events, including cytoskeletally-mediated cell spreading and tyrosine phosphorylation of focal adhesion kinase, pp125FAK, fail to occur. These studies establish the importance of the membrane-distal portion of the integrin beta3 cytoplasmic domain in bidirectional transmembrane signaling in human platelets, and the role of integrin signaling in maintaining normal hemostasis in vivo.

Amino Acid Sequence↗
PubMed2003

Mechanisms of hepatocyte growth factor-mediated vascular smooth muscle cell migration.

The migration of vascular smooth muscle cells (SMCs) from the media into the neointima and their subsequent proliferation is important in the pathogenesis of atherosclerosis. This process is regulated by multiple factors, including growth factors, and involves changes in the interaction of SMCs with the extracellular matrix and in intracellular signaling cascades that regulate cell movement. We demonstrated previously that hepatocyte growth factor (HGF) is expressed in human atherosclerotic plaques. Although HGF has been shown to promote SMC migration, the mechanisms involved in this process have not been characterized fully. In this study, inhibitory antibodies were used to determine which integrins mediated HGF-induced SMC migration. Inhibition of beta1 or beta3 integrin resulted in a significant decrease in migration. Subsequent experiments were performed to characterize additional biochemical mechanisms involved in HGF-mediated migration. HGF induced the redistribution of focal adhesions, the activation of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) and their increased association with beta1 and beta3 integrins, and the production of pro-matrix metalloproteinase-2. Migration levels were significantly reduced by cotreatment of SMCs with the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, UO126, the p38 inhibitor, SB203580, or the phosphatidylinositol-3 kinase inhibitor, LY294002. In HGF-treated SMCs, focal adhesion redistribution and FAK and Pyk2 activation were decreased by ERK1/2 inhibition. Neither SB203580 nor LY294002 inhibited HGF-induced ERK1/2 activation. Thus, ERK1/2 signaling may play an important role in HGF-mediated SMC migration by contributing to focal adhesion redistribution and FAK and Pyk2 activation.

Animals↗
PubMed1998

NF-kappaB mediates alphavbeta3 integrin-induced endothelial cell survival.

The alphavbeta3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-kappaB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and beta3 integrin ligation rapidly increased NF-kappaB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a beta1-integrin ligand) did not induce NF-kappaB activity. The alphavbeta3 integrin was most important for osteopontin-mediated NF-kappaB induction and survival, since adding a neutralizing anti-beta3 integrin antibody blocked NF-kappaB activity and induced endothelial cell death when cells were plated on osteopontin. NF-kappaB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-kappaB activity with nonphosphorylatable IkappaB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-kappaB was not required for fibronectin, laminin, and collagen type I-induced survival. Activation of NF-kappaB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-kappaB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-kappaB activation. These studies identify NF-kappaB as an important signaling molecule in alphavbeta3 integrin-mediated endothelial cell survival.

Animals↗
PubMed2005

Therapeutic expression of the platelet-specific integrin, alphaIIbbeta3, in a murine model for Glanzmann thrombasthenia.

Integrins mediate the adhesion of cells to each other and to the extracellular matrix during development, immunity, metastasis, thrombosis, and wound healing. Molecular defects in either the alpha- or beta-subunit can disrupt integrin synthesis, assembly, and/or binding to adhesive ligands. This is exemplified by the bleeding disorder, Glanzmann thrombasthenia (GT), where abnormalities of the platelet-specific integrin, alphaIIbbeta3, prevent platelet aggregation following vascular injury. We previously used a retrovirus vector containing a cDNA cassette encoding human integrin beta3 to restore integrin alphaIIbbeta3 on the surface of megakaryocytes derived from peripheral blood stem cells of GT patients. In the present study, bone marrow from beta3-deficient (beta3-/-) mice was transduced with the ITGbeta3-cassette to investigate whether the platelet progeny could establish hemostasis in vivo. A lentivirus transfer vector equipped with the human ITGA2B gene promoter confined transgene expression to the platelet lineage. Human beta3 formed a stable complex with murine alphaIIb, effectively restoring platelet function. Mice expressing significant levels of alphaIIbbeta3 on circulating platelets exhibited improved bleeding times. Intravenous immunoglobulin effectively diminished platelet clearance in animals that developed an antibody response to alphaIIbbeta3. These results indicate the feasibility of targeting platelets with genetic therapies for better management of patients with inherited bleeding disorders.

Animals↗
PubMed2006

Nanoparticle imaging of integrins on tumor cells.

Nanoparticles 10 to 100 nm in size can deliver large payloads to molecular targets, but undergo slow diffusion and/or slow transport through delivery barriers. To examine the feasibility of nanoparticles targeting a marker expressed in tumor cells, we used the binding of cyclic arginine-glycine-aspartic acid (RGD) nanoparticle targeting integrins on BT-20 tumor as a model system. The goals of this study were: 1) to use nanoparticles to image alpha(V)beta3 integrins expressed in BT-20 tumor cells by fluorescence-based imaging and magnetic resonance imaging, and, 2) to identify factors associated with the ability of nanoparticles to target tumor cell integrins. Three factors were identified: 1) tumor cell integrin expression (the alpha(V)beta3 integrin was expressed in BT-20 cells, but not in 9L cells); 2) nanoparticle pharmacokinetics (the cyclic RGD peptide cross-linked iron oxide had a blood half-life of 180 minutes and was able to escape from the vasculature over its long circulation time); and 3) tumor vascularization (the tumor had a dense capillary bed, with distances of <100 microm between capillaries). These results suggest that nanoparticles could be targeted to the cell surface markers expressed in tumor cells, at least in the case wherein the nanoparticles and the tumor model have characteristics similar to those of the BT-20 tumor employed here.

Animals↗