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Results for “Integrin alphaVbeta3”

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PubMed2005

ADAM15 decreases integrin alphavbeta3/vitronectin-mediated ovarian cancer cell adhesion and motility in an RGD-dependent fashion.

We have recently described that integrin alphavbeta3 upon interaction with its major extracellular matrix ligand vitronectin induces adhesion, motility, and proliferation of human ovarian cancer cells. Due to the important function of alphavbeta3 in cancer cell biology, it has been the effort of many scientific approaches to specifically target alphavbeta3-mediated cell adhesion and tumorbiological effects arising thereof by synthetic integrin antagonists. More recently, proteins of the ADAM family have been recognized as naturally occurring integrin ligands. Among those, human ADAM15 which encompasses the integrin binding RGD motif was shown to interact with integrin alphavbeta3. Thus, we investigated in human ovarian OV-MZ-6 cancer cells, expressing both ADAM15 and alphavbeta3, whether ADAM15 might affect alphavbeta3-mediated tumorbiological effects. We stably (over)expressed ADAM15 or its extracellular domain in OV-MZ-6 cells as well as respective ADAM15 mutants containing the tripeptide SGA instead of RGD. Cells (over)expressing ADAM15-RGD exhibited a significantly reduced alphavbeta3-mediated adhesion to vitronectin. Also, a significant time-dependent decline in numbers of cells cultivated on vitronectin was noticed. This effect was found to be rather due to impaired alphavbeta3-mediated cell adhesion than decreased cell proliferation rates, since de novo DNA synthesis was not significantly altered by elevated ADAM15 expression. Moreover, a substantially decreased random cellular motility was noticed as a function of ADAM15 encompassing an intact RGD motif. In conclusion, our results point to a physiological role of ADAM15 as a natural binding partner of integrin alphavbeta3 thereby loosening tumor cell adhesion to the underlying matrix and regulating tumor cell migration and invasion.

ADAM Proteins↗

PubMed2002

Osteopontin gene expression and immunolocalization in the rabbit urinary tract.

PURPOSE: Osteopontin is a highly phosphorylated, calcium binding sialoprotein characterized by a conserved arginine-glycine-aspartate sequence. Vitronectin receptor (alphavbeta3 integrin) and hyaluronan receptor (CD44) are documented as receptors for osteopontin and their expression has been established in the bladder. Based on that finding and the fact that osteopontin protein is present in urine we hypothesized that osteopontin is expressed in the lower urinary tract. MATERIALS AND METHODS: Osteopontin messenger (m)RNA and protein were analyzed in 5 adult urinary tracts and 5 neonatal bladders of New Zealand White rabbits using reverse transcriptase-polymerase chain reaction and immunohistochemical testing. Analysis of mRNA expression and localization of osteopontin receptors, alphavbeta3 integrin and CD44 were also performed in adult bladders and primary cultures of detrusor myocytes. RESULTS: Adult renal pelvis, ureter, bladder and urethra, and neonatal bladders contained significant levels of osteopontin mRNA. Immunohistochemical staining revealed osteopontin expression in all layers of the transitional epithelium of the bladder, co-localizing with alphavbeta3 integrin mainly in the superficial layers and with CD44 mainly in the basal layers. Osteopontin was detected within the cytoplasm of smooth muscle cells, while alphavbeta3 integrin was located closer to the plasmalemma. Furthermore, primary cultured detrusor myocytes expressed osteopontin mRNA in stable fashion for up to 4 passages. Treating bladder myocyte cultures with insulin-like growth factor-1 and 17beta-estradiol resulted in up-regulation and down-regulation of osteopontin mRNA, respectively. CONCLUSIONS: Adult and neonatal rabbit detrusors are a prominent source of osteopontin in vivo and in vitro. Epithelial osteopontin may be a source of osteopontin in urine. The co-localization of osteopontin in the bladder epithelium with alphavbeta3 integrin and CD44 suggests a role in maintaining the integrity of the transitional epithelium by providing the sealing and adhesiveness needed for the impermeable state of the bladder.

Animals↗

PubMed2003

The effect of different hormone therapies on integrin expression and pinopode formation in the human endometrium: a controlled study.

BACKGROUND: Integrin expression and pinopode formation have been proposed as a means of distinguishing receptive endometrium from non-receptive in clinical practice, thus offering new directions for the development of contraceptive approaches targeted to the endometrium as well as a better understanding of occult causes of infertility in women. The aim of the present study was to investigate the effect of ovulation induction, oral contraception, treatment with dehydrogesterone, and different regimens of hormone replacement therapy on endometrial alphavbeta3 integrin expression and pinopode formation using a prospective, controlled study design. METHODS: Histological dating, alphavbeta3 integrin expression and pinopode formation were evaluated in control and treated cycles in six groups of women including eight subjects per group and who received clomiphene citrate, ovarian stimulation for IVF, oral contraception, dehydrogesterone for endometrial luteal phase defect, or two different regimens of hormone replacement therapy. Twelve healthy fertile women served as a general control group. RESULTS: Alphavbeta3 integrin expression and pinopode formation in the human endometrium were closely related to endometrial maturation as defined by histological dating and this was irrespective of endometria being in-phase or out-of-phase and the hormonal treatment received. Only for clomiphene citrate did a direct effect with reduction in pinopode formation in the midluteal phase seem to exist. CONCLUSION: Alphavbeta3 integrin expression and pinopode formation in the human endometrium are processes closely related to endometrial maturation and this is irrespective of endometria being in-phase or out-of-phase and the hormonal treatment received. The potential usefulness of those two so-called endometrial markers of implantation as targets for contraceptive approaches or fertility-promoting strategies seems unlikely.

Adult↗

PubMed1999

Decreased angiogenesis and arthritic disease in rabbits treated with an alphavbeta3 antagonist.

Rheumatoid arthritis (RA) is an inflammatory disease associated with intense angiogenesis and vascular expression of integrin alphavbeta3. Intra-articular administration of a cyclic peptide antagonist of integrin alphavbeta3 to rabbits with antigen-induced arthritis early in disease resulted in inhibition of synovial angiogenesis and reduced synovial cell infiltrate, pannus formation, and cartilage erosions. These effects were not associated with lymphopenia or impairment of leukocyte function. Furthermore, when administered in chronic, preexisting disease, the alphavbeta3 antagonist effectively diminished arthritis severity and was associated with a quantitative increase in apoptosis of the angiogenic blood vessels. Therefore, angiogenesis appears to be a central factor in the initiation and persistence of arthritic disease, and antagonists of integrin alphavbeta3 may represent a novel therapeutic strategy for RA.

Animals↗

PubMed2000

Functional activation of integrin alpha V beta 3 in tumor cells expressing membrane-type 1 matrix metalloproteinase.

Matrix metalloproteinases (MMPs) and integrins have been implicated in a variety of processes involved in tumor progression. To evaluate the individual roles of integrin alphavbeta3 and membrane-type 1 matrix metalloproteinase (MT1-MMP), as well as the effects of their joint expression on tumor cell functions, MCF7 breast carcinoma cells were transfected stably with either the MT1-MMP, the beta3 integrin subunit or both MT1-MMP and beta3 cDNAs. MT1-MMP expression is accompanied by the functional activation of integrin alphaVbeta3, thereby increasing vitronectin-mediated adhesion and migration of MCF7 cells transfected with MT1-MMP and integrin alphaVbeta3. MT1-MMP-dependent functional activation of alphaVbeta3 correlates with modification(s) of the beta3 subunit, including its higher electrophoretic mobility and affected the LM609-binding site. MCF7 cells jointly expressing MT1-MMP and alphaVbeta3 were the most efficient in adhesion to the recombinant C-terminal domain of MMP-2 as well as in generating soluble and cell surface associated mature MMP-2 enzyme. These findings suggest a mechanism of selective docking of MMP-2 at tumor cell surfaces, specifically at the sites that include MT1-MMP and activated integrin alphaVbeta3. These mechanisms may provide a link between spatial regulation of focal proteolysis by the cell surface associated MMPs and the regulation of integrin-mediated motility of tumor cells.

Antigens, CD↗

PubMedDate not supplied

Endometrial microvascular growth in normal and dysfunctional states.

As a tissue that exhibits rapid cyclical growth and shedding throughout the reproductive life of the female, human endometrium provides a good model for the study of normal physiological angiogenesis. The objective of this paper is to summarize recent data on endometrial vascular growth, present new data on regional variability in endothelial cell proliferation within the endometrium, and interpret this information in light of current knowledge of the mechanisms by which angiogenesis occurs. Conventional angiogenesis normally involves a series of steps which include endothelial cell activation, breakdown of the basement membrane, migration and proliferation of the endothelial cell, fusion of sprouts, and tube formation. Other mechanisms by which angiogenesis occurs include intussusception and vessel elongation. Using immunohistochemical techniques we have shown repeatedly that levels of endothelial cell proliferation within human endometrium do not show any consistent pattern across the different stages of the menstrual cycle, which is unexpected since significant vascular growth must occur during the proliferative phase, when the endometrium increases in thickness by up to 4-fold. There are two possible explanations for this; either there is no obligatory link between endometrial endothelial cell proliferation and new vessel formation, or there is significant variation in endothelial cell proliferation within different regions of the same uterus. Multiple samples from hysterectomy specimens subsequently demonstrated that the variability is due to real differences between individuals, as well as showing that the endothelial cell proliferation index is significantly elevated in functionalis compared with basalis. During these studies we observed that endothelial cell proliferation nearly always appeared inside existing endometrial vessels, rather than be associated with structures that could be identified as vascular sprouts. To explore further whether sprout formation occurs during endometrial angiogenesis, we investigated the immunohistochemical distribution of integrin alphavbeta3 on endometrial endothelial cells. As for endothelial cell proliferation, integrin alphavbeta3 immunostaining was seen only on endothelial cells that appeared within existing blood vessels. The results from these studies have major implications for our understanding of the mechanisms that control endometrial angiogenesis. The lack of correlation between menstrual cycle stage and endothelial cell proliferation index, or endothelial cell expression of integrin alphavbeta3, suggests that vascular growth is not under the overall control of oestrogen and progesterone.

Animals↗

PubMed2006

A conjugate of camptothecin and a somatostatin analog against prostate cancer cell invasion via a possible signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9.

Camptothecin (CPT) was conjugated to the N-terminal of a somatostatin analog (SSA) directly via a carbamate group and a basic N-terminal linking motif, D-Lys-D-Tyr-Lys-D-Tyr-D-Lys. This new CPT-SSA conjugate termed JF-10-81 was evaluated as a receptor-specific delivery system for its anti-invasive and anti-angiogenic activities. It was found that, in addition to blocking migration and invasion of highly invasive prostate cancer PC-3 cells, this conjugate also inhibited in vitro capillary-like tube formation of endothelial cells and in vivo angiogenesis in C57B1/6N female mice. JF-10-81 was found to block PC-3 cell attachment to various extracellular matrix components, mainly to vitronectin, the ligand of cell surface receptors integrin alphaVbeta3 and alphaVbeta5. Additionally, JF-10-81 reduced expression of integrins alphaVbeta3 and alphaVbeta5 on PC-3 cell surfaces, without effects on beta1 or any alphabeta1 heterodimers. This conjugate also inactivated phosphorylation of protein kinase B (PKB/Akt), down-regulated the expression of latent matrix metalloproteinase (MMP) -2 and MMP-9, but had little effect on MMP-3/-10. Meanwhile, membrane type-1 matrix metalloproteinase (MT1-MMP) and the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were not detectable in PC-3 cells. alphaVbeta3/alphaVbeta5 and MMP-2/-9 are known to be highly expressed in many tumor cells and play an important role in tumor progression. Our results support that this conjugate could possibly inhibit prostate cancer PC-3 cell invasion through a signaling pathway involving PI3K/Akt, alphaVbeta3/alphaVbeta5 and MMP-2/-9, and this SSA could be used as an efficient vector to deliver CPT or other cytotoxic agents to target sites for cancer therapy.

Amino Acid Sequence↗

PubMed1999

Role of endothelial nitric oxide synthase in endothelial cell migration.

Endothelium-derived nitric oxide (NO) and its precursor L-arginine have been implied to promote angiogenesis, but little is known about the precise mechanism. The inhibition of endogenous NO formation by Nomega-nitro-L-arginine methyl ester (L-NAME) (1 mmol/L) but not its inactive enantiomer D-NAME (1 mmol/L) inhibited endothelial cell sprouting from the scratched edge of the cultured bovine aortic endothelial cell monolayer. Inhibition of endogenous NO release by L-NAME was confirmed by amperometric measurement using an NO-specific electrode. In the modified Boyden chamber, L-NAME (1 mmol/L) significantly inhibited endothelial cell migration, whereas L-NAME did not affect endothelial DNA synthesis as assessed by analysis of [3H]thymidine incorporation. We then examined alteration of endothelial cell adhesion molecule expression after the inhibition of NO by L-NAME in cultured human umbilical vein endothelial cells. In both normoxic and hypoxic conditions, L-NAME (1 mmol/L) inhibited surface expression of integrin alphavbeta3, which is an important integrin facilitating endothelial cell survival and angiogenesis. However, L-NAME did not affect the expression of platelet endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, vascular endothelial adhesion molecule-1, gap junction protein connexin 43, and VE-cadherin, which have been reported to potentially affect angiogenesis. In summary, inhibition of endothelial NO synthase by L-NAME attenuated endothelial cell migration but not proliferation in vitro. Furthermore, endogenous endothelium-derived NO maintains the functional expression of integrin alphavbeta3, a mediator for endothelial migration, survival, and angiogenesis. Endothelium-derived NO, thus, may play an important role in mediating angiogenesis by supporting endothelial cell migration, at least partly, via an integrin-dependent mechanism.

Animals↗

PubMed2006

MR three-dimensional molecular imaging of intramural biomarkers with targeted nanoparticles.

In this study, porcine carotid arteries were subjected to balloon overstretch injury followed by local delivery of paramagnetic nanoparticles targeted to alphavbeta3-integrin expressed by smooth muscle cells or collagen III within the extracellular matrix. Carotid T1-weighted angiography and vascular imaging was performed at 1.5T. While MR angiograms were indistinguishable between control and targeted vessel segments, alphavbeta3-integrin-and collagen Ill-targeted nanoparticles spatially delineated patterns and volumes of stretch injury. In conclusion, MR molecular imaging with alphavbeta3-integrin or collagen Ill-targeted nanoparticles enables the non-invasive, three-dimensional characterization of arterial pathology unanticipated from routine angiography.

Animals↗

PubMed2006

Integrin-linked kinase regulates osteopontin-dependent MMP-2 and uPA expression to convey metastatic function in murine mammary epithelial cancer cells.

Metastasis-supporting physiological alterations are regulated by cell signaling molecules, which target signal transduction pathways and gene expression. Osteopontin (OPN) overexpression may represent a key molecular event in cancer metastasis. In this study, using metastatic 4T1 and non-metastatic 4T07 murine mammary cancer cell lines, we demonstrate that 4T1 cells exhibit significantly increased OPN, integrin-linked kinase (ILK), matrix metalloproteinase-2 (MMP-2) and urokinase-type plasminogen activator (uPA) expression in contrast to 4T07 cells. Blockade of OPN binding to 4T1 cell-surface integrins by the competitive ligand inhibitor, RGD, or a blocking antibody to alphavbeta3 integrin decreases OPN, ILK, MMP-2 and uPA expression. Conversely, exposure of 4T07 cells to exogenous OPN increases ILK, MMP-2 and uPA levels. Further experiments demonstrate that OPN-alphavbeta3 integrin binding in 4T1 with subsequent activation of ILK results in binding of AP-1 to MMP-2 and uPA promoter and increased in vitro promoter activation, as measured by transient transfection assays using MMP-2 and uPA promoter-reporter constructs. AP-1 activity is ablated by co-transfection of DN-ILK or exposure to RGD. Finally, functional correlative assays demonstrate that inhibition of ILK activity or RGD-mediated blockade of alphavbeta3 integrin binding significantly inhibits in vitro invasion, migration and invasion properties of 4T1 cells. In addition, uPA and MMP-2 have overlapping contributions to 4T1 migration and invasion characteristics. However, OPN and ILK activities contribute to 4T1 adhesion activities via mechanisms that are independent of uPA and MMP-2. Our results indicate that binding of an RGD-bearing ligand, such as OPN, to integrin receptors in metastatic 4T1 cells transcriptionally mediates MMP-2, uPA and OPN expression through ILK-dependent AP-1 activity and significantly increases in vitro functional correlates of metastasis. In 4T1 murine mammary cancer cells, we conclude that OPN mediates metastatic behavior, in part, through upregulation of MMP-2 and uPA protein expression.

Animals↗

PubMed2005

Angiogenesis and the growth potential of craniopharyngiomas.

The present study was performed to evaluate the role of neovascularization on the behavior of craniopharyngiomas as well as the contribution of endothelial cell proliferation and migration in the remodeling and expansion of the vascular network associated with angiogenesis. Fourteen primary tumors were studied, all of the adamantinomatous type. CD34 immunostaining, an endothelial cell marker, localized vessels within the connective tissue stroma. MIB-1 immunopositivity was apparent in the nuclei of neoplastic cells, few endothelial cells, and stromal elements. MIB-1 counts were higher in epithelial than connective tissue cells. A positive correlation was found between the number of MIB-1 immunopositive cells and microvessel density (MVD). Immunohistochemistry demonstrated that integrin alphavbeta3 expression was restricted to tumor vasculature; the tumor cells were immunonegative. Only 2.5% of vessels detected with CD34 were immunopositive for integrin alphavbeta3. At present, no therapeutic implications can be drawn from our observations. More studies are needed to assess whether integrin alphavbeta3 antagonists or drugs that arrest the cell cycle of endothelial cells can inhibit angiogenesis in craniopharyngiomas.

Adolescent↗

PubMed2003

Association of sustained ERK activity with integrin beta3 induction during receptor activator of nuclear factor kappaB ligand (RANKL)-directed osteoclast differentiation.

Osteoclast differentiation is a multi-step process that involves cell proliferation, commitment, and fusion. Some adhesion molecules, including integrin alphavbeta3, have been shown to have roles in osteoclast fusion. In the course of studying with pharmacologic agents known to inhibit protein tyrosine kinases of the Src family, we found that radicicol increased cell fusion during receptor activator of nuclear factor kappaB ligand (RANKL)-driven differentiation of osteoclasts at concentrations far below the ones shown to inhibit its targets in previous studies. Treatments of low doses of radicicol to RAW 264.7 cells that undergo osteoclastic differentiation in the presence of RANKL enhanced the RANKL-induced gene expression of integrin beta3 without any effect on the expression of integrin alphav, which was constitutively high. The cell surface level of integrin alphavbeta3 complexes was consequently augmented by radicicol. In addition, sustained ERK and MEK activation was observed in cells treated with both radicicol and RANKL. More importantly, modulation of ERK activity by the MEK inhibitor U0126 or the gene transduction of a constitutively active form of MEK resulted in a suppression and increment, respectively, of integrin beta3 induction by RANKL. Our data indicate that sustained ERK activity is associated with integrin beta3 induction and subsequent cell surface expression of the alphavbeta3 integrin complex, which may contribute to cell fusion during RANKL-directed osteoclastogenesis.

Animals↗

PubMed2004

Osteopontin increases CD44 expression and cell adhesion in RAW 264.7 murine leukemia cells.

BACKGROUND: Osteopontin (OPN) is an inducible cell attachment protein which binds alphavbeta3-integrin and CD44 receptors to promote tumor metastasis. We hypothesized that OPN alters expression of its CD44 receptor to promote neoplastic cell migration. METHODS: RAW264.7 cells were stimulated with OPN (0-10 nM) for 0-12 hours to determine the time- and concentration-dependence of CD44 protein and mRNA expression. In selected instances, a competitive ligand for the alphavbeta3-integrin, GRGDSP (50 nM), or an inhibitor of protein synthesis, anisomycin (10 microg/ml), was added. Cell adhesion to hyaluronan was assayed with the crystal violet assay. RESULTS: OPN upregulates plasma membrane total CD44 protein in a concentration-(ANOVA P = 0.001) and time-dependent (ANOVA P = 0.001) fashion. CD44v6 is not altered. Cell adhesion to hyaluronate increases in parallel with CD44 expression. Steady state mRNA levels for CD44 are not altered by OPN. 5 nM OPN increases CD44 protein half-life from 105 +/- 11 minutes to 278 +/- 15 minutes. (P < 0.03) Blockade of either alphavbeta3-integrin ablates the OPN-dependent increase in CD44. CONCLUSIONS: These data indicate that OPN increases plasma membrane CD44 expression and cell adhesion by binding to its alphavbeta3-integrin receptor. We conclude that OPN may promote tumor metastatic behavior by CD44 expression.

Animals↗

PubMed2001

Monoclonal antibody-mediated drug targeting to choroidal neovascularization in the rat.

PURPOSE: Active drug targeting mediated by monoclonal antibodies (mAbs) of vascular endothelial cells in tumors is a new concept in cancer therapy. Integrin alphavbeta3 has been reported to be strongly expressed in vascular endothelial cells of surgically excised choroidal neovascular membranes and is thought to be a potential antigen for mAb-mediated drug targeting of choroidal neovascularization (CNV). The objective of this study was to evaluate the efficacy of drug targeting mediated by anti-integrin alphavbeta3 mAbs in a laser-induced CNV rat model. METHODS: The mitomycin C (MMC)-dextran (MMCD) conjugate was synthesized with a carbodiimide-catalyzed reaction. The mAb was conjugated with MMCD (MMCD-mAb). To evaluate the feasibility of mAb-mediated drug targeting in vitro, we investigated the effect of the immunoconjugates involving dextran-binding MMC on the proliferation of human umbilical vein endothelial cells (HUVECs). CNV was induced by laser photocoagulation in male Brown Norway rats. Immunolocalization of integrin alphavbeta3 in CNV lesions was assessed immunohistochemically with the anti-von Willebrand factor antibody as an endothelial cell marker. Intravenous administration of saline (n = 7), 1 mg/day mAb (n = 7), 100 microg/kg per day free MMC (n = 7), MMCD with irrelevant Ab (n = 7), unconjugated MMCD with unconjugated mAb (MMCD+mAb; n = 7), or MMCD with mAb (MMCD-mAb; n = 8) containing an equal amount of free MMC, was performed daily for 3 days from day 14 after CNV induction. CNV was assessed by fluorescein angiography 2 weeks after treatment. Fluorescein leakage was scored on a four-grade scale. The animals were killed 2 weeks after treatment, and the lesions were evaluated histologically. RESULTS: The inhibition of immunoconjugates on the proliferation of HUVECs was enhanced specifically by the mediatory effect of the mAb. Endothelial cells demonstrated strong immunoreactivity of integrin alphavbeta3 in the CNV. In the vehicle-treated group, fluorescein leakage equal to that before treatment was observed 2 weeks after treatment, with an average score of 2.00 +/- 0.17 (mean +/- SEM). MMCD-mAb significantly inhibited the development of CNV in rats (P < 0.01). Moreover, the thickness of the lesions was significantly reduced in the MMCD-mAb-treated group (P < 0.01). CONCLUSIONS: Immunoconjugates effectively inhibited progression of CNV in this model. The results suggest that mAb-mediated drug targeting may be beneficial in the treatment of CNV.

Animals↗

PubMed2006

The adhesion molecule L1 (CD171) promotes melanoma progression.

The adhesion molecule L1 is expressed in primary melanomas and cutaneous metastases in contrast to melanocytic nevi and melanocytes, and is significantly associated with metastatic spread. Recent studies have demonstrated that in carcinomas L1 expression is associated with sustained activation of the extracellular signal-regulated kinase (ERK) pathway and upregulation of ERK-dependent, motility- and invasion-associated gene products including alphavbeta3 integrin. The objective of this study was to further investigate the role of the adhesion molecule L1 in melanoma progression, and to evaluate whether targeting the L1 adhesion molecule would have therapeutic effects against invasive melanoma growth. Using human melanoma cells from different stages of progression in monolayer and organotypic human skin culture mimicking the pathophysiological environment of cutaneous melanoma, we found that (1) L1 expression mostly correlates with melanoma progression and alphavbeta3 integrin expression, (2) overexpression of L1 in early radial growth phase melanoma cells promotes conversion from radial to vertical growth phase melanoma without upregulation of alphavbeta3 integrin expression, and (3) suppression of L1 function significantly reduces migration and invasion of melanoma cells, but does not completely block invasive melanoma growth. Altogether, L1 plays a critical role in melanoma invasion and progression and offers therapeutic potential in combination with conventional anticancer agents.

Animals↗

PubMed2003

Hepatocyte growth factor activates endothelial proangiogenic mechanisms relevant in chronic hepatitis C-associated neoangiogenesis.

BACKGROUND: Angiogenesis occurs in inflamed portal tracts of chronic hepatitis C (CHC) patients. AIMS: To characterize this phenomenon, by investigating the molecular mechanisms involved in neovessel formation in the livers of CHC patients and the angiogenic effects of hepatocyte growth factor (HGF) on human endothelial cells. METHODS: Vascular endothelial growth factor (VEGF), VE-cadherin and alphavbeta3 integrin were determined in CHC biopsies by Western blot and immunohistochemistry. Effects of HGF on VEGF and cell adhesion molecules expression by cultured human microvascular endothelial cells were evaluated by Western blot, Northern blot or immunofluorescence. HGF effects on cell proliferation were assessed by [(3)H]thymidine incorporation. RESULTS: VEGF, VE-cadherin and alphavbeta3 integrin were increased in CHC liver samples. In cultured endothelial cells, HGF transcriptionally increased VEGF expression, an effect which was blocked by an anti-VEGF receptor antibody. HGF transiently decreased VE-cadherin expression and its associated cytoskeleton-linking molecule beta-catenin, thus weakening intercellular contacts. HGF increased alphavbeta3 integrin at focal contacts, and cell proliferation, an effect which was inhibited by an anti-VEGF receptor antibody. CONCLUSIONS: Our results show that HGF and VEGF modulate the expression of cell adhesion and migration molecules and induce proliferation in endothelial cells, mechanisms through which these factors may contribute to CHC-associated liver angiogenesis.

Adult↗

PubMed2006

Polymeric conjugates of mono- and bi-cyclic alphaVbeta3 binding peptides for tumor targeting.

The alphaVbeta3 integrin plays important roles in tumor-induced angiogenesis and tumor metastasis and hence, many small molecule alphaVbeta3 ligands have been developed for cancer diagnosis and therapy. Although these show good alphaVbeta3 targeting, most have suboptimal pharmacokinetics and show rapid tumor washout. We studied the biodistribution and tumor targeting properties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer based conjugates of mono-(RGDfK) and doubly cyclized (RGD4C) alphaVbeta3 binding peptides. Endothelial cell adhesion studies showed similar affinity of HPMA-RGD4C and HPMA-RGDfK conjugate for alphaVbeta3 integrins. Scintigraphic images of tumor bearing mice demonstrated that both conjugates showed tumor localization at 24 h post-injection and were retained at the tumor site until 192 h, whereas the efficient background clearance was observed over time. Necropsy organ counts showed that tumor accumulation of both HPMA-RGD4C and HPMA-RGDfK conjugates increased over time with peak accumulations at 4.9 +/- 0.9% and 5.0 +/- 1.2% ID/g, respectively. In contrast the background organ distribution rapidly cleared over time resulting in significant increases of tumor-to-background ratios. The radioactive dose as indicated by the area under curve (HPMA-RGD4C: 4825.3 microCi/g h and HPMA-RGDfK: 4424.9 microCi/g h) was highest for the tumor. The polymer conjugates of RGD4C or RGDfK provide a means to enhance tumor uptake, decrease background accumulation, and enable selective delivery of therapeutic or diagnostic agents to tumor sites.

Amino Acid Sequence↗

PubMed2006

Down-regulation of uPAR and cathepsin B retards cofilin dephosphorylation.

Cathepsin B and uPAR play key roles in cancer cell migration and invasion. Here, we demonstrate that the simultaneous, siRNA-mediated down-regulation of uPAR and cathepsin B inhibits glioma cell migration and is accompanied by cytoskeletal condensation. We show that the dephosphorylation of cofilin is inhibited by the down-regulation of uPAR alone and, to a lesser extent, by the down-regulation of cathepsin B alone, and that the effect was much higher with the down-regulation of both molecules by pUC. Using FACS analysis and western blotting for the alphaVbeta3 integrin heterodimer, we determined that down-regulating uPAR subsequently causes the down-regulation of the alphaVbeta3 integrin heterodimer. As evidenced by western blot analysis of ERK1/2, pERK1/2, p38MAPK, p-p38MAPK, AKT, pAKT and PI3-k, the MEK and PI3-k pathways are inhibited. From cytoskeleton studies, we observed that the down-regulation of uPAR caused cytoskeletal condensation and that the simultaneous down-regulation of uPAR and cathepsin B was even more effective at inducing cytoskeletal condensation than uPAR alone. Our results demonstrate the relevance of uPAR in cytoskeletal dynamics and the potential of uPAR and cathepsin B as targets in the treatment of malignant gliomas.

Actin Depolymerizing Factors↗