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PubMed2002

Role of integrin alpha(v)beta3 in the early phase of liver metastasis: PET and IVM analyses.

To clarify the function of integrin alpha(v)beta3 in the early stage of liver metastasis, we investigated the interactions of metastatic cells with their target organ under the actual blood flow by using positron emission tomography (PET). The cells used were CHO-K1 cells and their transfectants bearing human integrin alpha(v)beta3 cDNA (alpha(v)beta3-CHO-K1 cells). The liver accumulation of alpha(v)beta3-CHO-K1 cells was significantly higher than that of CHO-K1 cells after injection via the portal vein, whereas no significant difference was observed in the lung accumulation after tail vein injection, suggesting a specific interaction of alpha(v)beta3-CHO-K1 cells with the hepatic sinusoids. Furthermore, to clarify the precise location of each cell in the liver, i.e., to determine whether individual cells were intravascularly localized or had extravasated, we performed intravital fluorescence microscopy (IVM) on the liver by using stable transfectants bearing the green fluorescent protein (GFP) gene, namely, GFP-CHO-K1 and GFP-alpha(v)beta3-CHO-K1 cells. Both types of cells remained in the hepatic blood vessels 1 h after injection via the portal vein. On the other hand, expression of integrin alpha(v)beta3 promoted the cells to reach the extravascular region after 24 h. These results suggest the possibility that the specific accumulation of alpha(v)beta3-CHO-K1 cells in the liver is followed by migration of the cells into the extravascular region. Interestingly, the adhesion of the two types of cells to hepatic sinusoidal endothelial cells in vitro did not correspond to in vivo accumulation of these cells. Therefore, integrin alpha(v)beta3 may function to promote extravasation of integrin alpha(v)beta3-expressing tumor cells in liver through a process possibly mediated by vitronectin produced by this organ.

Animals↗
PubMed2003

Localization of integrin alpha(v)beta3 and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in cutaneous and oral melanomas of dog.

Melanomas are common neoplasms of dogs and arise from pigment-producing cells called melanocytes or melanoblasts. Melanomas of skin are often easily cured by surgical excision, but those of oral mucosa are aggressive, metastasize to the regional lymph nodes and lungs, and respond poorly to conventional therapy. Tumor growth is sustained by proliferation of microvessels via a process called angiogenesis. Integrin alpha(v)beta3 is expressed in proliferating but not in quiescent microvessels suggesting a role in angiogenesis. Vascular endothelial growth factor (VEGF) manifests its mitogenic and angiogenic effects mainly via VEGF receptor-2 (VEGFR-2/Flk-1). We conducted this immunocytochemical study to investigate the expression of integrin alpha(v)beta3 and VEGFR-2 in archival and fresh samples from cases of canine melanomas. Results show that integrin alpha(v)beta3 was expressed in 72% and 88% of cutaneous and oral melanomas, respectively, and the expression was restricted to and immediately around the melanocytes and endothelial cells. VEGFR-2 staining of selected cases of melanoma revealed that its expression overlapped with the alpha(v)beta3 integrin. Dual immuno-gold electron microscopy confirmed co-localization of integrin alpha(v)beta3 and VEGFR-2 in melanocytes and endothelial cells. These data demonstrate expression and co-localization of integrin alpha(v)beta3 and VEGFR-2 in cutaneous and oral melanomas of dogs.

Animals↗
PubMed2004

Thromboxane A2 receptor agonists antagonize the proangiogenic effects of fibroblast growth factor-2: role of receptor internalization, thrombospondin-1, and alpha(v)beta3.

Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of the pathogenesis of many vascular events, including angiogenesis. Endothelial cells express TXA2 receptors (TP) but the effects of TP stimulation on angiogenesis remain controversial. In this study, we show that stimulation of endothelial cell TP impairs ligand-induced FGF receptor internalization and consequently abrogates FGF-2-induced endothelial cell migration in vitro and angiogenesis in vivo. Prevention of FGF-2-induced angiogenesis was associated with expression of the TPbeta isoform. The deficit in FGFR1 internalization was mediated through activation of TPbeta preventing the FGF-2-mediated decrease in p53 expression, thus enhancing thrombospondin-1 (TSP-1) release from EC and reducing FGFR1 internalization. Once released TSP-1 interacted with the alpha(v)beta3 integrin on the EC surface. On stimulation, FGFR1 and alpha(v)beta3 were found to associate in a complex. We determined that complex formation was important for receptor internalization as conditions that inhibit FGFR1 internalization, such as inappropriate ligation of alpha(v)beta3 by either TSP-1 or a neutralizing antibody, disrupted the complex. These results establish a novel role for isoform specific regulation of angiogenesis by TP, provide the first functional significance for the existence of two TP isoforms in humans, and clarify the mechanism by which TP signaling regulates FGFR1 kinetics and signaling.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed2004

Transmembrane domain helix packing stabilizes integrin alphaIIbbeta3 in the low affinity state.

Regulated changes in the affinity of integrin adhesion receptors ("activation") play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the alpha and beta transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to alpha/beta interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the beta3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the alpha and beta TM domains. Therefore, we propose that helical packing of the alpha and beta TM domains forms a clasp that regulates integrin activation.

Amino Acid Sequence↗
PubMed1998

cAMP does not inhibit convulxin-induced tyrosyl-phosphorylation of human platelet proteins, including PLCgamma2, but completely blocks the integrin alphaIIb beta3-dependent dephosphorylation step: comparisons with RGDS peptide, cytochalasin D, and phenylarsine oxide.

Convulxin (Cvx) isolated from Crotalus durissus terrificus venom, induces platelet aggregation, phospholipase C (PLC) activation, and tyrosyl-phosphorylation (PTP) of multiple proteins, including PLCgamma2 by a mechanism independent of integrin alphaIIb beta3. However, PTP induced by Cvx is followed by a dephosphorylation step in a platelet aggregation-dependent manner. Here we show that increasing intraplatelet content of cAMP with forskolin is associated with the inhibition of Cvx-induced platelet aggregation, ATP secretion, and inositol-phosphates production. However, the early onset of Cvx-induced PTP is not sensitive to cAMP (including PLCgamma2), and it also occurs in the presence of integrin alphaIIb beta3-antagonist (RGDS peptide, RGDS) or inhibitors of actin polymerization (cytochalasin D, CD) and tyrosine-phosphatases (phenylarsine oxide, PAO). However, forskolin, RGDS, and CD prevented the dephosphorylation step together with inhibition of platelet aggregation, whereas in the presence of phenylarsine oxide (PAO) the dephosphorylation step was replaced by an increase in the number and intensity of tyrosyl-phosphorylated proteins. Our data provide evidence to conclude that (i) cAMP inhibits platelet aggregation at a downstream site to PLCgamma2 tyrosyl-phosphorylation; (ii) Cvx-induced PTP is independent on integrin alphaIIb beta3 engagement, actin polymerization, and tyrosine-phosphatases activation; (iii) integrin alphaIIb beta3 mediates the dephosphorylation step in a platelet aggregation-dependent manner; and (iv) Cvx and collagen stimulate platelets by a similar signal transduction pathway.

Arsenicals↗
PubMed2005

Radiolabeled RGD uptake and alphav integrin expression is enhanced in ischemic murine hindlimbs.

UNLABELLED: Radiolabeled RGD peptides that target alpha(v)beta3 integrin are promising tracers for imaging tumor angiogenesis. Integrins and angiogenesis also play important roles in healing of ischemic lesions. Thus, we investigated the biodistribution of radiolabeled RGD and expression of alpha(v) integrin in a mouse model of hindlimb ischemia. METHODS: 125I-3-Iodo-D-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) (125I-c(RGD(I)yV)) was synthesized and tested for endothelial binding. Hindlimb ischemia was induced in ICR mice through femoral artery ablation, and perfusion was measured with laser Doppler blood flowmetry. 125I-c(RGD(I)yV) biodistribution was evaluated in control animals (n = 7) and ischemic models on day 3, 8, or 14 (n = 6 each). Control experiments were performed using a radiolabeled peptide with a scrambled amino acid sequence (125I-GfVGV). Microsections of hindlimb tissue were immunostained for alpha(v) integrin expression and stained with alkaline phosphatase to localize vascular endothelial cells. RESULTS: 125I-c(RGD(I)yV) retained specific binding to human umbilical vein endothelial cells. Perfusion in ischemic hindlimbs immediately fell to 10% +/- 4% of contralateral levels and gradually recovered to 22% +/- 11% and 64% +/- 9% on days 8 and 14, respectively. 125I-c(RGD(I)yV) uptake in ischemic muscles significantly increased from a control level of 0.16 +/- 0.05 %ID/g (percentage injected dose per gram of tissue) to 0.85 +/- 0.76 %ID/g at day 3, 0.43 +/- 0.23 %ID/g at day 8, and 0.43 +/- 0.28 %ID/g at day 14 (all P < 0.05). Ischemic muscle-to-lung count ratios had a virtually identical trend: 0.42 +/- 0.25 for controls, 2.34 +/- 1.70 at day 3 (P < 0.02), 1.46 +/- 0.52 at day 8 (P < 0.001), and 1.39 +/- 0.94 at day 14 (P < 0.02). In contrast, uptake of the control peptide in ischemic hindlimbs was not different from that of controls. Immunohistochemistry revealed substantially increased alpha(v) integrin staining in ischemic hindlimb tissue. CONCLUSION: Radioiodine RGD uptake is significantly enhanced in ischemic hindlimbs of a mouse model, and is accompanied by an increase in alpha(v) integrin expression. Further investigation is thus warranted to illuminate the potential role of radiolabeled RGD for noninvasive monitoring of peripheral ischemic lesions.

Animals↗
PubMed2002

Integrin alpha(v)beta3 targeted therapy for Kaposi's sarcoma with an in vitro evolved antibody.

Here, we define integrin alpha(v)beta3 as a molecular target for antibody therapy for Kaposi's sarcoma (KS). We previously reported, using a new phage display strategy based on designed combinatorial V gene libraries, the humanization of mouse monoclonal antibody LM609 directed to human integrin alpha(v)beta3. In the present study, we describe the in vitro affinity maturation of humanized LM609 by using a phage display strategy for the sequential and parallel optimization of three complementarity determining regions of the antibody molecule. The evolved Fab had an affinity of 150 pM and was converted into IgG1 by use of a new mammalian expression vector. The resulting whole antibody, designated JC-7U IgG1, was found to selectively target human KS in a nude mouse model and inhibit tumor growth at a therapeutically relevant dose. Because of its high affinity and its high degree of humanization, JC-7U IgG1 is an excellent drug candidate for therapeutic applications that involve integrin alpha(v)beta3 as the molecular target. Of particular interest is therapy for KS, breast cancer, melanoma, and other cancers in which integrin alpha(v)beta3 is expressed on both angiogenic endothelial cells and tumor cells, which would allow a dual antiangiogenic and antitumor strike with a single drug.

Animals↗
PubMed2004

PKD1/PKCmu promotes alphavbeta3 integrin recycling and delivery to nascent focal adhesions.

To identify kinases that regulate integrin recycling, we have immunoprecipitated alphavbeta3 integrin from NIH 3T3 fibroblasts in the presence and absence of primaquine (a drug that inhibits receptor recycling and leads to accumulation of integrins in endosomes) and screened for co-precipitating kinases. Primaquine strongly promoted association of alphavbeta3 integrin with PKD1, and fluorescence microscopy indicated that integrin and PKD1 associate at a vesicular compartment that is downstream of a Rab4-dependent transport step. PKD1 association was mediated by the C-terminal region of the beta3 integrin cytodomain, and mutants of beta3 that were unable to recruit PKD1 did not recycle in a PDGF-dependent fashion. Furthermore, suppression of endogenous PKD1 levels by RNAi, or overexpression of catalytically inactive PKD1 inhibited PDGF-dependent recycling of alphavbeta3 from early endosomes to the plasma membrane and blocked recruitment of alphavbeta3 to newly formed focal adhesions during cell spreading. These data indicate that PKD1 influences cell migration by directing vesicular transport of the alphavbeta3 integrin heterodimer.

Animals↗
PubMed1999

Definition of an unexpected ligand recognition motif for alphav beta6 integrin.

Integrin interactions with extracellular matrix proteins are mediated by brief oligopeptide recognition sequences, and synthetic peptides containing such sequences can inhibit integrin binding to the matrix. The RGD peptide motif is recognized by many integrins including alphav beta6, a specific receptor for fibronectin thought to support epithelial cell proliferation during wound healing and carcinoma progression. We report here the discovery of an unexpected non-RGD recognition motif for integrin alphav beta6. We compared the recognition profiles of recombinant alphav beta6 and alphav beta3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to alphav beta3 contained ubiquitous RGD sequences. However, on alphav beta6, in addition to RGD- containing phages, one-quarter of the population from the 12-mer library contained the distinctive consensus motif DLXXL. A synthetic DLXXL peptide, RTDLDSLRTYTL, selected from the phage sequences (clone-1) was a selective inhibitor of RGD-dependent ligand binding to alphav beta6 in isolated receptor assays (IC50 = 20 nM), and in cell adhesion assays (IC50 = 50 microM). DLXXL peptides were highly specific inhibitors of alphav beta6-fibronectin interaction as synthetic scrambled or reversed DLXXL peptides were inactive. NH2- and COOH-terminal modifications of the flanking amino acids suggested that the preceding two and a single trailing amino acid were also involved in interaction with alphav beta6. The DLXXL sequence is present in several matrix components and in the beta chain of many integrins. Although there is as yet no precise biological role known for DLXXL, it is clearly a specific inhibitory sequence for integrin alphav beta6 which has been unrecognized previously.

Amino Acid Sequence↗
PubMed1998

Foot-and-mouth disease virus virulent for cattle utilizes the integrin alpha(v)beta3 as its receptor.

Adsorption and plaque formation of foot-and-mouth disease virus (FMDV) serotype A12 are inhibited by antibodies to the integrin alpha(v)beta3 (A. Berinstein et al., J. Virol. 69:2664-2666, 1995). A human cell line, K562, which does not normally express alpha(v)beta3 cannot replicate this serotype unless cells are transfected with cDNAs encoding this integrin (K562-alpha(v)beta3 cells). In contrast, we found that a tissue culture-propagated FMDV, type O1BFS, was able to replicate in nontransfected K562 cells, and replication was not inhibited by antibodies to the endogenously expressed integrin alpha5beta1. A recent report indicating that cell surface heparan sulfate (HS) was required for efficient infection of type O1 (T. Jackson et al., J. Virol. 70:5282-5287, 1996) led us to examine the role of HS and alpha(v)beta3 in FMDV infection. We transfected normal CHO cells, which express HS but not alpha(v)beta3, and two HS-deficient CHO cell lines with cDNAs encoding human alpha(v)beta3, producing a panel of cells that expressed one or both receptors. In these cells, type A12 replication was dependent on expression of alpha(v)beta3, whereas type O1BFS replicated to high titer in normal CHO cells but could not replicate in HS-deficient cells even when they expressed alpha(v)beta3. We have also analyzed two genetically engineered variants of type O1Campos, vCRM4, which has greatly reduced virulence in cattle and can bind to heparin-Sepharose columns, and vCRM8, which is highly virulent in cattle and cannot bind to heparin-Sepharose. vCRM4 replicated in wild-type K562 cells and normal, nontransfected CHO (HS+ alpha(v)beta3-) cells, whereas vCRM8 replicated only in K562 and CHO cells transfected with alpha(v)beta3 cDNAs. A similar result was also obtained in assays using a vCRM4 virus with an engineered RGD-->KGE mutation. These results indicate that virulent FMDV utilizes the alpha(v)beta3 integrin as a primary receptor for infection and that adaptation of type O1 virus to cell culture results in the ability of the virus to utilize HS as a receptor and a concomitant loss of virulence.

Animals↗
PubMed2005

PI3-kinase is essential for ADP-stimulated integrin alpha(IIb)beta3-mediated platelet calcium oscillation, implications for P2Y receptor pathways in integrin alpha(IIb)beta3-initiated signaling cross-talks.

Phosphatidylinositol 3-kinase (PI3K) pathway is important for platelet activation. Recent studies showed that PI3K and oscillative calcium could cross talk to each other and positively regulate integrin alpha (IIb)beta3-mediated outside-in signaling. However, the mechanism of this feedback regulation remains to be further characterized. Here we found that treatments of both PI3K inhibitor wortmannin and P2Y1 inhibitor A3P5P could inhibit granular secretion in platelets. Additionally, when RGD-substrate adherent platelets were treated with the ADP scavenger apyrase to deplete the granular-released ADP, their attachments in engaging with substrates became looser and the frequency of calcium oscillation decreased. Since it is known that ADP stimulates the PI3K and calcium signal primarily through P2Y12 and P2Y1 receptors respectively, our data indicated that integrin alpha(IIb)beta3 downstream PI3K and calcium activation might be not completely coupled to integrin associated signaling complex, but in part through feedback stimulation by granular released ADP. Our data indicates the important roles of PI3K and granular released ADP in coordinating the feedback regulations in integrin alpha(IIb)beta3-mediated platelet activation.

Actins↗
PubMed1998

CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize beta1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion.

Cell migration may depend on integrin-mediated adhesion to and deadhesion from extracellular matrix ligands. This concept, however, has not yet been confirmed for T lymphocytes migrating in three-dimensional extracellular matrices. We investigated receptor involvement in T cell migration combining a three-dimensional collagen matrix model with time-lapse videomicroscopy, computer-assisted cell tracking and confocal microscopy. In collagen lattices, the migration of CD4+ T cells (1) involved interactions with collagen fibers at the leading edge and uropod likewise, (2) occurred independently of the co-clustering of beta1, beta2, or beta3 integrins with F-actin, focal adhesion kinase, and phosphotyrosine at interactions with collagen fibers, (3) was counteracted by high-affinity beta1 integrin binding induced by antibody TS2/16; however, (4) the migration could not be blocked by a combination of adhesion-perturbing anti-beta1, -beta2, -beta3, and alpha v integrin antibodies. Integrin blocking neither affected cell polarization, interaction with fibers, beta1 integrin distribution, migration velocity, path structure, nor the number of locomoting cells in spontaneously migrating or concanavalin A-activated cells. Hence, T lymphocytes migrating in three-dimensional collagen matrices may utilize highly transient interactions with collagen fibers of low adhesivity, thereby differing from focal adhesion-dependent migration strategies employed by other cells.

Antibodies, Monoclonal↗
PubMed2006

JAM-C regulates tight junctions and integrin-mediated cell adhesion and migration.

Junctional Adhesion Molecules (JAMs) have been described as major components of tight junctions in endothelial and epithelial cells. Tight junctions are crucial for the establishment and maintenance of cell polarity. During tumor development, they are remodeled, enabling neoplastic cells to escape from constraints imposed by intercellular junctions and to adopt a migratory behavior. Using a carcinoma cell line we tested whether JAM-C could affect tight junctions and migratory properties of tumor cells. We show that transfection of JAM-C improves the tight junctional barrier in tumor cells devoid of JAM-C expression. This is dependent on serine 281 in the cytoplasmic tail of JAM-C because serine mutation into alanine abolishes the specific localization of JAM-C in tight junctions and establishment of cell polarity. More importantly, the same mutation stimulates integrin-mediated cell migration and adhesion via the modulation of beta1 and beta3 integrin activation. These results highlight an unexpected function for JAM-C in controlling epithelial cell conversion from a static, polarized state to a pro-migratory phenotype.

Amino Acid Sequence↗
PubMed2006

Redox regulation of 4-hydroxy-2-nonenal-mediated endothelial barrier dysfunction by focal adhesion, adherens, and tight junction proteins.

4-Hydroxy-2-nonenal (4-HNE), one of the major biologically active aldehydes formed during inflammation and oxidative stress, has been implicated in a number of cardiovascular and pulmonary disorders. 4-HNE has been shown to increase vascular endothelial permeability; however, the underlying mechanisms are unclear. Hence, in the current study, we tested our hypothesis that 4-HNE-induced changes in cellular thiol redox status may contribute to modulation of cell signaling pathways that lead to endothelial barrier dysfunction. Exposure of bovine lung microvascular endothelial cells (BLMVECs) to 4-HNE induced reactive oxygen species generation, depleted intracellular glutathione, and altered cell-cell adhesion as measured by transendothelial electrical resistance. Pretreatment of BLM-VECs with thiol protectants, N-acetylcysteine and mercaptopropionyl glycine, attenuated 4-HNE-induced decrease in transendothelial electrical resistance, reactive oxygen species generation, Michael protein adduct formation, protein tyrosine phosphorylation, activation of ERK, JNK, and p38 MAPK, and actin cytoskeletal rearrangement. Treatment of BLMVECs with 4-HNE resulted in the redistribution of FAK, paxillin, VE-cadherin, beta-catenin, and ZO-1, and intercellular gap formation. Western blot analyses confirmed the formation of 4-HNE-derived Michael adducts with the focal adhesion and adherens junction proteins. Also, 4-HNE decreased tyrosine phosphorylation of FAK without affecting total cellular FAK contents, suggesting the modification of integrins, which are natural FAK receptors. 4-HNE caused a decrease in the surface integrin in a time-dependent manner without altering total alpha5 and beta3 integrins. These results, for the first time, revealed that 4-HNE in redox-dependent fashion affected endothelial cell permeability by modulating cell-cell adhesion through focal adhesion, adherens, and tight junction proteins as well as integrin signal transduction that may lead dramatic alteration in endothelial cell barrier dysfunction during heart infarction, brain stroke, and lung diseases.

Adherens Junctions↗
PubMed2005

Role of angiotensin type-1 and angiotensin type-2 receptors in the expression of vascular integrins in angiotensin II-infused rats.

Angiotensin II plays an important role in vascular remodeling through effects that involve, in part, interactions of vascular smooth muscle cells with extracellular matrix via integrins, which belong to a family of transmembrane receptors. We hypothesized that angiotensin (Ang) II regulates expression of vascular integrins and their ligands in experimental hypertension. Rats were infused subcutaneously with Ang II and received angiotensin type-1 (AT1) receptor blocker losartan, the AT1/angiotensin type-2 (AT2) [Sar1-Ile8]-Ang II, or the vasodilator hydralazine for 7 days. Osteopontin and integrin subunit expression were evaluated immunohistochemically. Ang II enhanced vascular alpha8, beta1, beta3 integrins and osteopontin expression, which were significantly reduced by losartan, [Sar1-Ile8]-Ang II, and hydralazine. Although Ang II increased vascular alpha5 subunit expression, this was additionally increased by losartan. Losartan was the only treatment that induced alpha1 subunit expression. These results demonstrate that AT1 and AT2 receptors have countervailing effects on vascular integrin subunit expression that may influence their effects on vascular remodeling and extracellular matrix composition.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed2006

Platelet-derived growth factor BB-mediated normalization of dermal interstitial fluid pressure after mast cell degranulation depends on beta3 but not beta1 integrins.

Interstitial fluid pressure (PIF) is one of the determinants of transcapillary fluid flux and thereby interstitial fluid volume. Cell-mediated control of PIF regulates fluid content in the loose interstitial connective tissues that surround the capillary bed. To maintain a normal PIF in dermis, beta1 integrins mediate the tensile strength applied by connective tissue cells on the extracellular matrix. Platelet-derived growth factor (PDGF)-BB normalizes anaphylaxis-induced reduction of PIF. Anti-beta3 integrin IgG and a cyclic RGD peptide that inhibits the alphaVbeta3 integrin blocked the ability of PDGF-BB to normalize the lowered PIF resulting from mast cell degranulation. PDGF-BB was unable to normalize PIF lowered as a result of mast cell degranulation in beta3-negative mice. Monoclonal anti-beta3 integrin IgG had no effect on PIF in normal mouse dermis. In contrast, administration of anti-beta1 integrin IgM lowered PIF in normal dermis but had no effect on PDGF-BB-induced normalization of PIF after anaphylaxis. Furthermore, collagen gel contraction mediated by wild-type mouse embryonal fibroblasts were only marginally affected by function-blocking anti-beta1 integrin antibodies, especially in the presence of PDGF-BB. In contrast, contraction mediated by alphaV-negative mouse embryonic fibroblasts was completely blocked by anti-beta1 integrin antibodies, even after stimulation with PDGF-BB. These results show a previously unrecognized in vivo function for the alphaVbeta3 integrin, as a participant in the control of PIF during inflammatory reactions. Furthermore, our data demonstrate that PDGF-BB induces connective tissue cells to generate tensile forces via alphaVbeta3 during such reactions.

Animals↗
PubMed2006

PET-based human dosimetry of 18F-galacto-RGD, a new radiotracer for imaging alpha v beta3 expression.

UNLABELLED: (18)F-Galacto-RGD is a new tracer for PET imaging of alpha v beta3, a receptor involved in a variety of pathologic processes including angiogenesis and metastasis. Our aim was to study the dosimetry of (18)F-galacto-RGD in humans. METHODS: Eighteen patients with various tumors (musculoskeletal tumors [n = 10], melanoma [n = 5], breast cancer [n = 2], or head and neck cancer [n = 1]) were examined. After injection of 133-200 MBq of (18)F-galacto-RGD, 3 consecutive emission scans from the thorax to the pelvis were acquired at 6.7 +/- 2.9, 35.6 +/- 7.6, and 70.4 +/- 12.2 min after injection. Blood samples (n = 4) for metabolite analysis were taken 10, 30, and 120 min after injection. The OLINDA 1.0 program was used to estimate the absorbed radiation dose. RESULTS: Reversed-phase high-performance liquid chromatography of serum revealed that more than 95% of tracer was intact up to 120 min after injection. (18)F-Galacto-RGD showed rapid clearance from the blood pool and primarily renal excretion. Background activity in lung and muscle tissue was low (percentage injected dose per liter at 71 min after injection, 0.56 +/- 0.15 and 0.69 +/- 0.25, respectively). The calculated effective dose was 18.7 +/- 2.4 microSv/MBq, and the highest absorbed radiation dose was in the bladder wall (0.22 +/- 0.03 mGy/MBq). CONCLUSION: (18)F-Galacto-RGD demonstrates high metabolic stability, a favorable biodistribution, and a low radiation dose. Consequently, this tracer can safely be used for noninvasive imaging of molecular processes involving the alpha v beta3 integrin and for the planning and monitoring of therapeutic approaches targeting alpha v beta3.

Adult↗
PubMed2005

BAP31 and its caspase cleavage product regulate cell surface expression of tetraspanins and integrin-mediated cell survival.

BAP31, a resident integral protein of the endoplasmic reticulum membrane, regulates the export of other integral membrane proteins to the downstream secretory pathway. Here we show that cell surface expression of the tetraspanins CD9 and CD81 is compromised in mouse cells from which the Bap31 gene has been deleted. CD9 and CD81 facilitate the function of multiprotein complexes at the plasma membrane, including integrins. Of note, BAP31 does not appear to influence the egress of alpha5beta1 or alpha(v)beta3 integrins to the cell surface, but in Bap31-null mouse cells, these integrins are not able to maintain cellular adhesion to the extracellular matrix in the presence of reduced serum. Consequently, Bap31-null cells are sensitive to serum starvation-induced apoptosis. Reconstitution of wild-type BAP31 into these Bap31-null cells restores integrin-mediated cell attachment and cell survival after serum stress, whereas interference with the functions of CD9, alpha5beta1, or alpha(v)beta3 by antagonizing antibodies makes BAP31 cells act similar to Bap31-null cells in these respects. Finally, in human KB epithelial cells protected from apoptosis by BCL-2, the caspase-8 cleavage product, p20 BAP31, inhibits egress of tetraspanin and integrin-mediated cell attachment. Thus, p20 BAP31 can operate upstream of BCL-2 in living cells to influence cell surface properties due to its effects on protein egress from the endoplasmic reticulum.

Animals↗