Biomedical subjects

D A Cheresh

Publications and source records attributed to D A Cheresh.

At least 19 recordsLinked to original sources

PubMed2001

Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins.

Integrin-mediated adhesion promotes cell survival in vitro, whereas integrin antagonists induce apoptosis of adherent cells in vivo. Here, we demonstrate that cells adherent within a three-dimensional extracellular matrix undergo apoptosis due to expression of unligated integrins, the beta subunit cytoplasmic domain, or its membrane proximal sequence KLLITIHDRKEF. Integrin-mediated death requires initiator, but not stress, caspase activity and is distinct from anoikis, which is caused by the loss of adhesion per se. Surprisingly, unligated integrin or beta integrin tails recruit caspase-8 to the membrane, where it becomes activated in a death receptor-independent manner. Integrin ligation disrupts this integrin-caspase containing complex and increases survival, revealing an unexpected role for integrins in the regulation of apoptosis and tissue remodeling.

Animals↗

PubMed2001

Identification of a novel class of small-molecule antiangiogenic agents through the screening of combinatorial libraries which function by inhibiting the binding and localization of proteinase MMP2 to integrin alpha(V)beta(3).

The process of new blood vessel growth from existing vasculature, known as angiogenesis, is critical to several pathological conditions, most notably cancer. Both MMP2, which degrades the extracellular matrix (ECM), and integrin alpha(V)beta(3), which contributes to endothelial cell attachment to the ECM, are critically involved in this process. Recent findings have shown that MMP2 is localized in an active form on the surface of invasive endothelial cells based on its ability to directly bind integrin alpha(V)beta(3), suggesting that disrupting this protein--protein interaction may represent a new target for the development of angiogenesis inhibitors. The screening of small molecule libraries led to the identification of compounds which disrupt the MMP2--alpha(V)beta(3) interaction in an in vitro binding assay. A prototypical inhibitor was further found to prevent the degradation of the protein matrix without directly inhibiting MMP2 activity or disrupting the binding of alpha(V)beta(3) to its classical ECM ligand, vitronectin. The synthesis and screening of analogues and substructures of this lead compound allowed the identification of requisite structural features for inhibition of MMP2 binding to alpha(V)beta(3). This led to the synthesis of a more water-soluble derivative which maintains the in vitro biological properties and has potent antiangiogenic and antitumor activity in vivo, validating the target as one useful for therapeutic intervention.

Angiogenesis Inhibitors↗

PubMed2001

Disruption of matrix metalloproteinase 2 binding to integrin alpha vbeta 3 by an organic molecule inhibits angiogenesis and tumor growth in vivo.

Matrix metalloproteinase 2 (MMP2) can associate with integrin alpha(v)beta3 on the surface of endothelial cells, thereby promoting vascular invasion. Here, we describe an organic molecule (TSRI265) selected for its ability to bind to integrin alphav(v)beta3 and block alpha(v)beta3 interaction with MMP2. Although disrupting alpha(v)beta3/MMP2 complex formation, TSRI265 has no effect on alpha(v)beta3 binding to its extracellular matrix ligand vitronectin and does not influence MMP2 activation or catalytic activity directly. However, TSRI265 acts as a potent antiangiogenic agent and thereby blocks tumor growth in vivo. These findings suggest that activated MMP2 does not facilitate vascular invasion during angiogenesis unless it forms a complex with alpha(v)beta(3) on the endothelial cell surface. By disrupting endothelial cell invasion without broadly suppressing cell adhesion or MMP function, the use of compounds such as TSRI265 may provide a novel therapeutic approach for diseases associated with uncontrolled angiogenesis.

Angiogenesis Inhibitors↗

PubMed2001

Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging.

Viral vectors based on lentiviruses, such as the human immunodeficiency virus, are able to transduce a broad spectrum of nondividing cells in vivo. This ability of lentiviral vectors makes them an attractive vehicle for gene transfer into the liver. In order to determine the requirements for efficient lentiviral gene transfer, we used a fluorescence imaging system, which allows the detection of cells and tissues that express fluorescent reporter genes (e.g., green fluorescence protein) in the living animal. We show that the latest generation of lentiviral vectors efficiently transduces the murine liver. Further analysis demonstrated that neither cell-cycle activation nor division of liver cells is a prerequisite for lentiviral gene transfer in vivo.

Animals↗

PubMed2001

Adhesion events in angiogenesis.

Recent work from several laboratories indicates that the coordination of endothelial cell adhesion events with growth factor receptor inputs regulates endothelial cell responses during angiogenesis. Analyses of the signaling pathways downstream of integrins, cadherins and growth-factor receptors are providing an insight into the molecular basis of known anti-angiogenic strategies, as well as into the design of novel therapies.

Angiogenesis Inhibitors↗

PubMed2001

Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke.

Vascular endothelial growth factor (VEGF), an angiogenic factor produced in response to ischemic injury, promotes vascular permeability (VP). Evidence is provided that Src kinase regulates VEGF-mediated VP in the brain following stroke and that suppression of Src activity decreases VP thereby minimizing brain injury. Mice lacking pp60c-src are resistant to VEGF-induced VP and show decreased infarct volumes after stroke whereas mice deficient in pp59c-fyn, another Src family member, have normal VEGF-mediated VP and infarct size. Systemic application of a Src-inhibitor given up to six hours following stroke suppressed VP protecting wild-type mice from ischemia-induced brain damage without influencing VEGF expression. This was associated with reduced edema, improved cerebral perfusion and decreased infarct volume 24 hours after injury as measured by magnetic resonance imaging and histological analysis. Thus, Src represents a key intermediate and novel therapeutic target in the pathophysiology of cerebral ischemia where it appears to regulate neuronal damage by influencing VEGF-mediated VP.

Animals↗

PubMed2001

Preferential susceptibility of brain tumors to the antiangiogenic effects of an alpha(v) integrin antagonist.

OBJECTIVE: Brain tumors are highly angiogenic, and their growth and spread depend on the generation of new blood vessels. We examined the effect of the cyclic peptide antagonist pentapeptide EMD 121974, an antiangiogenic agent, on orthotopic and heterotopic brain tumor growth. METHODS: The human brain tumor cell lines DAOY (medulloblastoma) and U87 MG (glioblastoma) were injected into either the forebrain (orthotopic) or the subcutis (heterotopic) of nude mice, and daily systemic treatment with the active peptide was initiated after tumors were established. RESULTS: All control animals with orthotopic brain tumors and that received the inactive peptide EMD 135981 daily died as a result of tumor progression within 4 to 6 weeks; tumors measured 3 to 5 mm in diameter. In contrast, mice with orthotopic tumors that were treated daily with the active peptide survived for more than 16 weeks, and histological examination of the brains after 4, 8, and 12 weeks showed either no tumors or microscopic residual tumors. The growth of these brain tumor cells injected simultaneously or separately into the subcutis of nude mice (heterotopic model) was not affected by the active peptide, suggesting that the brain environment is a critical determinant of brain tumor susceptibility to growth inhibition by this pentapeptide. CONCLUSION: The cyclic pentapeptide EMD 121974 may become a treatment option specific to brain tumors. Because of its antiangiogenic effect, its use may be especially indicated after tumors are removed surgically.

Allantois↗

PubMed2001

Alpha(v)beta3 and alpha(v)beta5 integrin expression in glioma periphery.

OBJECTIVE: This study analyzed the expression of integrins alpha(v)beta3 and alpha(v)beta5 in glioma tissue and focused on the periphery of high-grade gliomas. METHODS: The analysis was performed with Western blot, immunohistochemistry, and immunofluorescence, by use of two monoclonal antibodies able to recognize the functional integrin heterodimer. The expression of integrin-related ligands and growth factors also was studied. Sections from the tumor periphery were classified as either tumor periphery (light tumor infiltrate or scant visible cells) or peritumor (heavy tumor infiltration). RESULTS: Our data on glioma tissues demonstrated that both integrins were expressed in glioma cells and vasculature and their expression correlated with the histological grade. Alpha(v)beta3 expression was prominent in astrocytic tumors. Both integrins were markers of tumor vasculature, particularly of endothelial proliferation. A high-grade glioma periphery demonstrated a prominent expression of integrin alpha(v)beta3. Cells demonstrating alpha(v)beta3 positivity were identified as tumor astrocytes and endothelial cells by double imaging. The same cells were surrounded by some alpha(v)beta3 ligands and co-localized fibroblast growth factor 2. Matrix metalloproteinase 2 also was found to be co-localized with alpha(v)beta3 in the same cells. Alpha(v)beta3 expression was more relevant in tumor astrocytes. Alpha(v)beta3 integrin and vascular endothelial growth factor expression increased from the periphery to the tumor center. CONCLUSION: Our data support the role of integrins alpha(v)beta3 and alpha(v)beta5 in glioma-associated angiogenesis. In addition, they suggest a role for integrin alpha(v)beta3 in neoangiogenesis and cell migration in high-grade glioma periphery.

Adult↗

PubMed2001

Integrin alpha(v)beta1 is an adenovirus coreceptor.

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.

Adenoviridae↗

PubMed2000

Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic fragment of matrix metalloproteinase 2.

Modulation of the balance between pro- and antiangiogenic factors holds great promise for the treatment of a broad spectrum of human disease ranging from ischemic heart disease to cancer. This requires both the identification of angiogenic regulators and their efficient delivery to target organs. Here, we demonstrate the use of a noncatalytic fragment of matrix metalloproteinase 2 (termed PEX) delivered by lentiviral vectors in different angiogenesis models. Transduction of human endothelial cells with PEX virus suppressed endothelial invasion and formation of capillary-like structures without affecting chemotaxis in vitro. Lentiviral delivery of PEX blocked basic fibroblast growth factor-induced matrix metalloproteinase 2 activation and angiogenesis on chicken chorioallantoic membranes. PEX expression also inhibited tumor-induced angiogenesis and tumor growth in a nude mouse model. Thus, our study shows that lentiviral vectors can deliver sufficient quantities of antiangiogenic substances to achieve therapeutic effects in vivo.

Animals↗

PubMed2000

beta1 integrin antagonism on adherent, differentiated human neuroblastoma cells triggers an apoptotic signaling pathway.

Integrin receptors mediate several functions including prevention of matrix detachment-induced apoptosis (anoikis) of several adherent cell types. We report here that antagonists of beta1 integrins trigger an apoptotic signaling pathway in adherent differentiated LAN-5 human neuroblastoma cells, a cell line which represents a model system for the study of human neurons. The pathway is characterized by cytochrome c release into the cytoplasm, and activation of caspase-9 and caspase-3, 4-6h after treatment; cleavage products of caspase-8 and caspase-2 were not detectable in the cells. Coordinate inactivation of cell survival pathways, including cleavage of focal adhesion kinase, decreased expression of protein kinase B, and reduced phosphorylation of the pro-apoptotic protein, Bad, also characterized the signaling pathway. These events occurred in adherent cells; DNA fragmentation and detachment followed as late events 18-24h after addition of beta1 integrin antagonists. zDEVD-fmk, an irreversible inhibitor of caspase-3-like enzymes, and cytochalasin D, an actin depolymerizing agent, blocked caspase-3 cleavage and delayed cell death. In contrast to these results, undifferentiated, adherent and dividing LAN-5 cells did not die in response to beta1 integrin antagonists. These studies identify a distinct apoptotic pathway which is triggered by antagonists of beta1 integrins on differentiated adherent neuronal cells.

Antibodies, Monoclonal↗

PubMed2000

Alpha(v)beta3 and alpha(v)beta5 integrin expression in meningiomas.

OBJECTIVE: Integrins are emerging as alternative receptors capable of mediating several biological functions, such as cell-matrix and cell-cell adhesion, cell migration, signal transduction, and angiogenesis. Two alpha(v) integrins, i.e., alpha(v)beta3 and alpha(v)beta5, play critical roles in mediating these activities, particularly in tumors. No data are available on the expression of these integrins in meningiomas. METHODS: Using Western blot and immunohistochemical analyses with LM609 and PG32, two monoclonal antibodies capable of recognizing the functional integrin heterodimer, we evaluated the expression of alpha(v)beta3 and alpha(v)beta5 integrins in a series of 34 meningiomas of different histological subtypes and grades. We studied their expression in tumor cells and vasculature, as well as the expression of their related angiogenic factors (fibroblast growth factor 2 and vascular endothelial growth factor) and the alpha(v)beta3 ligand vitronectin. RESULTS: Alpha(v)beta3 and alpha(v)beta5 integrins were expressed by neoplastic vasculature and cells. Alpha(v)beta3 and alpha(v)beta5 expression was associated and correlated with that of their respective growth factors (fibroblast growth factor 2 and vascular endothelial growth factor) and microvessel counts and densities. Alpha(v)beta3 was more strongly expressed than alpha(v)beta5 in two cases of histologically benign meningiomas with aggressive clinical behavior. Alpha(v)beta3 expression was associated with that of its related ligand vitronectin and was also evident in small vessels of brain tissue closely surrounding meningiomas. CONCLUSION: Our data demonstrate the expression of alpha(v)beta3 and alpha(v)beta5 integrins in meningioma cells and vasculature. Our findings suggest a role for both of these integrins, and particularly alpha(v)beta3, in meningioma angiogenesis.

Adult↗

PubMed2000

Regulation of adenovirus membrane penetration by the cytoplasmic tail of integrin beta5.

Adenovirus (Ad) cell entry involves sequential interactions with host cell receptors that mediate attachment (CAR), internalization (alphavbeta3 and alphavbeta5), and penetration (alphavbeta5) of the endosomal membrane. These events allow the virus to deliver its genome to the nucleus. While integrins alphavbeta3 and alphavbeta5 both promote Ad internalization into cells, integrin alphavbeta5 selectively facilitates Ad-mediated membrane permeabilization and endosome rupture. In the experiments reported herein, we demonstrate that the intracellular domain of the integrin beta5 subunit specifically regulates Ad-mediated membrane permeabilization and gene delivery. CS-1 melanoma cells expressing a truncated integrin beta5 or a chimeric (beta5-beta3) cytoplasmic tail (CT) supported normal levels of Ad endocytosis but had reduced Ad-mediated gene delivery and membrane permeabilization relative to cells expressing a wild-type integrin beta5. Thin-section electron microscopy revealed that virion particles were capable of being endocytosed into cells expressing a truncated beta5CT, but they failed to escape cytoplasmic vesicles and translocate to the nucleus. Site-specific mutagenesis studies suggest that a C-terminal TVD motif in the beta5CT plays a major role in Ad membrane penetration.

Adenoviruses, Human↗

PubMed2000

Role of alpha v integrins during angiogenesis.

Angiogenesis depends on specific molecular interactions between vascular cells and components of the extracellular matrix. This review focuses on the recent advances in the understanding of the mechanism of action of integrins and integrin antagonists during angiogenesis. For example, angiogenesis induced with vascular endothelial growth/permeability factor but not with basic fibroblast growth factor (bFGF) depends on integrin avb5 and Src kinase activity. In contrast, bFGF-induced angiogenesis requires integrin avb3 and functions independently of Src. Recent studies document a role for integrins and growth factor regulation of Src family kinases during angiogenesis. We also discuss the effect of av integrin antagonists on angiogenesis during tumor growth, inflammatory disease, and retinopathy and summarize recent clinical progress in using av integrin antagonists.

Animals↗

PubMed2000

Targeted antiangiogenic therapy for cancer using Vitaxin: a humanized monoclonal antibody to the integrin alphavbeta3.

Angiogenesis plays a central role in the growth and metastasis of cancers. Strategies aimed at interfering with tumor blood supply offer promise for new cancer therapies. Vitaxin (an anti-alphavbeta3 antibody) interferes with blood vessel formation by inducing apoptosis in newly generated endothelial cells. This Phase I study evaluates the safety and pharmacokinetics of Vitaxin in humans with cancer. Eligible patients demonstrated progressive tumors with stage IV disease and an Eastern Cooperative Oncology Group performance status < or =2. Treatment consisted of six weekly infusions of Vitaxin. Escalating doses from 0.1 and 4.0 mg/kg/week were evaluated based on the expectation that plasma levels would bracket the effective in vitro concentration. Escalation beyond 4 mg/kg/week was limited by drug availability. Adverse events were assessed weekly. Pharmacokinetics were performed weekly through week 9. Clinical response was assessed at week 9. Of 17 patients treated, 14 were evaluable for response. Treatment was well tolerated with little or no toxicity. The most common side effect was infusion-related fever, which could be controlled with prophylactic antipyretics. Doses > or =1 mg/kg/week produced plasma concentrations sufficient to saturate the alphavbeta3 receptor in vitro (25 microg/ml). Vitaxin demonstrated a half-life in excess of 5 days at higher doses with no accumulation over 6 weeks of therapy. One patient demonstrated a partial response, and seven patients demonstrated stable disease. Three patients received Vitaxin beyond the first cycle of therapy. Each of these patients demonstrated disease stabilization that in one case lasted 22 months. At the doses and schedule studied, Vitaxin appears safe and potentially active, suggesting that vascular integrin alphavbeta3 represents a clinically relevant antiangiogenic target for prolonged cancer therapy.

Adult↗

PubMed1999

Potentiation of cell migration by adhesion-dependent cooperative signals from the GTPase Rac and Raf kinase.

The small GTPase Rac is thought to regulate cell movement by influencing actin cytoskeletal organization and membrane ruffling. However, cell migration also depends on the activation of mitogen-activated protein kinase (MAPK), which can regulate myosin motor function, an event critical for cell contraction. Evidence is provided that, during active cell adhesion to the extracellular matrix, Rac potentiates the MAPK pathway and influences cell migration by selectively synergizing with Raf kinase but not with Ras or MAPK kinase. In fact, the synergy between Rac and Raf kinase increases the chemotactic sensitivity of cells to epidermal growth factor by 1000-fold. Therefore, the role of Rac in cell migration not only depends on its ability to regulate actin cytoskeletal organization but also on its capacity to potentiate chemokine activation of MAPK in a manner that depends on active cell adhesion to the extracellular matrix.

3T3 Cells↗

PubMed1999

Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

Actins↗

PubMed1999

Mechanisms and consequences of affinity modulation of integrin alpha(V)beta(3) detected with a novel patch-engineered monovalent ligand.

Integrin alpha(V)beta(3) mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which alpha(V)beta(3) is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, alpha(IIb)beta(3), has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of alpha(V)beta(3), a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single alpha(V) integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated alpha(V)beta(3), but not to alpha(IIb)beta(3), in receptor and cell binding assays. alpha(V)beta(3) affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K(d) = 2.4 microM), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K(d) = 80 nM), with no change in receptor number. In contrast, alpha(V)beta(3) in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of beta(3) cytoplasmic tails. Up-regulation of alpha(V)beta(3) affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that alpha(V)beta(3) is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.

Adenoviridae↗