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Biomedical subjects

D A Cheresh

Publications and source records attributed to D A Cheresh.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1999

Integrin alpha(v)beta3 promotes M21 melanoma growth in human skin by regulating tumor cell survival.

Growth and dissemination of malignant melanoma has a profound impact on our population, and little is known concerning the mechanisms controlling this disease in humans. Evidence is provided that integrin alpha(v)beta3 plays a critical role in M21 melanoma tumor survival within human skin by a mechanism independent of its known role in angiogenesis. Antagonists of alpha(v)beta3 blocked melanoma growth by inducing tumor apoptosis. Moreover, M21 melanoma cell interactions with denatured collagen, a known ligand for alpha(v)beta3, caused a 5-fold increase in the relative Bcl-2:Bax ratio, an event thought to promote cell survival. Importantly, denatured collagen colocalized with alpha(v)beta3-expressing melanoma cells in human tumor biopsies, suggesting that alpha(v)beta3 interaction with denatured collagen may play a critical role in melanoma tumor survival in vivo.

Animals↗
PubMed1999

Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase.

Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.

Cell Adhesion↗
PubMed1999

Synergy between an antiangiogenic integrin alphav antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases.

The suppression and eradication of primary tumors and distant metastases is a major goal of alternative treatment strategies for cancer, such as inhibition of angiogenesis and targeted immunotherapy. We report here a synergy between two novel monotherapies directed against vascular and tumor compartments, respectively, a tumor vasculature-specific antiangiogenic integrin alphav antagonist and tumor-specific antibody-interleukin 2 (IL-2) fusion proteins. Simultaneous and sequential combination of these monotherapies effectively eradicated spontaneous liver metastases in a poorly immunogenic syngeneic model of neuroblastoma. This was in contrast to controls subjected to monotherapies with either an antiangiogenic integrin alphav antagonist or antibody-IL-2 fusion proteins, which were only partially effective at the dose levels applied. Furthermore, simultaneous treatments with the integrin alphav antagonist and tumor-specific antibody-IL-2 fusion proteins induced dramatic primary tumor regressions in three syngeneic murine tumor models, i.e., melanoma, colon carcinoma, and neuroblastoma. However, each agent used as monotherapy induced only a delay in tumor growth. A mechanism for this synergism was suggested because the antitumor response was accompanied by a simultaneous 50% reduction in tumor vessel density and a 5-fold increase in inflammatory cells in the tumor microenvironment. Subsequently, tumor necrosis was demonstrated only in animals receiving the combination therapy, but not when each agent was applied as monotherapy. The results suggest that these synergistic treatment modalities may provide a novel and effective tool for future therapies of metastatic cancer.

Animals↗
PubMed1999

Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability.

Src kinase activity was found to protect endothelial cells from apoptosis during vascular endothelial growth factor (VEGF)-, but not basic fibroblast growth factor (bFGF)-, mediated angiogenesis in chick embryos and mice. In fact, retroviral targeting of kinase-deleted Src to tumor-associated blood vessels suppressed angiogenesis and the growth of a VEGF-producing tumor. Although mice lacking individual Src family kinases (SFKs) showed normal angiogenesis, mice deficient in pp60c-src or pp62c-yes showed no VEGF-induced vascular permeability (VP), yet fyn-/- mice displayed normal VP. In contrast, inflammation-mediated VP appeared normal in Src-deficient mice. Therefore, VEGF-, but not bFGF-, mediated angiogenesis requires SFK activity in general, whereas the VP activity of VEGF specifically depends on the SFKs, Src, or Yes.

Animals↗
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↗
PubMed1999

A role for angiogenesis in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic debilitating disease characterized by distinct autoimmune, inflammatory and fibrovascular components which lead to synovial proliferation and joint destruction. However, existing treatments specifically target only autoimmune and inflammatory components despite the fact that neovascularization of the inflamed synovium is a hallmark of rheumatoid arthritis. Angiogenesis may contribute to synovial growth, leukocyte recruitment and tissue remodeling, thus potentiating disease progression. Although no therapies currently target angiogenesis, several existing therapies have anti-angiogenic activity. Recent advances in anti-angiogenic strategies in oncology, including the identification of integrin alpha v beta 3 as a crucial effector of angiogenesis, suggest a means to assess the role of angiogenesis in rheumatoid arthritis. Synovial endothelial cells have been shown to express integrin alpha v beta 3, suggesting that these cells may be targeted for angiogenesis inhibition. Prior studies in rat arthritis models have shown benefit after the addition of broad spectrum integrin antagonists. However, formal assessment of integrin-targeted anti-angiogenic activity is now underway. These controlled studies will be important in assessing the efficacy of therapies which target angiogenesis in RA.

Animals↗
PubMed1998

A phage display approach for rapid antibody humanization: designed combinatorial V gene libraries.

The development of a new strategy for antibody humanization is described. This strategy incorporates key recognition sequences from the parental rodent antibody into a phage display-based selection strategy. The original sequences of the third complementarity-determining regions (CDRs) of heavy and light chains, HCDR3 and LCDR3, were maintained and all other sequences were replaced by human sequences selected from phage-displayed antibody libraries. This approach was applied to the humanization of mouse mAb LM609 that is directed to human integrin alphav beta3 and has potential applicability in cancer therapy as an antiangiogenic agent. We demonstrate this approach (i) provides a rapid route for antibody humanization constraining the content of original mouse sequences in the final antibodies to the most hypervariable of the CDRs; (ii) generates several humanized versions with different sequences at the same time; (iii) results in affinities as high as or higher than the affinity of the original antibody; and (iv) retains the antigen and epitope specificity of the original antibody. The production of multiple humanized variants may present advantages in the selection of antibodies that are more readily expressed on a large scale and could be important in therapeutic regimens that call for long-term treatment with antibodies in which antiidiotypic responses might be avoided by administration of alternative antibodies.

Amino Acid Sequence↗
PubMed1998

Integrin alphavbeta3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis.

Angiogenesis depends on growth factors and vascular cell adhesion events. Integrins and growth factors are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic fibroblast growth factor (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least 20 h. The initial wave of ERK activity (5-120 min) was refractory to integrin antagonists, whereas the sustained activity (4-20 h) depended on integrin alphavbeta3, but not beta1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained alphavbeta3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked growth factor induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a growth factor receptor and integrin alphavbeta3.

Animals↗
PubMed1998

CAS/Crk coupling serves as a "molecular switch" for induction of cell migration.

Carcinoma cells selected for their ability to migrate in vitro showed enhanced invasive properties in vivo. Associated with this induction of migration was the anchorage-dependent phosphorylation of p130CAS (Crk-associated substrate), leading to its coupling to the adaptor protein c-CrkII (Crk). In fact, expression of CAS or its adaptor protein partner Crk was sufficient to promote cell migration, and this depended on CAS tyrosine phosphorylation facilitating an SH2-mediated complex with Crk. Cytokine-stimulated cell migration was blocked by CAS lacking the Crk binding site or Crk containing a mutant SH2 domain. This migration response was characterized by CAS/Crk localization to membrane ruffles and blocked by the dominant-negative GTPase, Rac, but not Ras. Thus, CAS/Crk assembly serves as a "molecular switch" for the induction of cell migration and appears to contribute to the invasive property of tumors.

Animals↗
PubMed1998

Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity.

Angiogenesis depends on both cell adhesion and proteolytic mechanisms. In fact, matrix metalloproteinase 2 (MMP-2) and integrin alphavbeta3 are functionally associated on the surface of angiogenic blood vessels. A fragment of MMP-2, which comprises the C-terminal hemopexin-like domain, termed PEX, prevents this enzyme binding to alphavbeta3 and blocks cell surface collagenolytic activity. PEX blocks MMP-2 activity on the chick chorioallantoic membrane where it disrupts angiogenesis and tumor growth. Importantly, a naturally occurring form of PEX can be detected in vivo in conjunction with alphavbeta3 expression in tumors and during developmental retinal neovascularization. Levels of PEX in these vascularized tissues suggest that it interacts with endothelial cell alphavbeta3 where it serves as a natural inhibitor of MMP-2 activity, thereby regulating the invasive behavior of new blood vessels.

Allantois↗
PubMed1998

Design, synthesis and biological evaluation of nonpeptide integrin antagonists.

Recent studies demonstrated that peptide and antibody antagonists of integrin alpha v beta 3 block angiogenesis and tumor growth. In this article, the design, synthesis and biological evaluation of a series of nitroaryl ether-based, nonpeptide mimetics are described. The design of these compounds was based on Merck's arylether/alpha-aminoacid/guanidine framework and incorporates a novel nitroaryl system. The synthesized mimetics were tested against a variety of integrins (alpha v beta 3, alpha IIb beta 3, and alpha v beta 5) in order to determine their binding selectivity and ability to inhibit cell adhesion. Selected compounds were also tested for their ability to inhibit angiogenesis in vivo in the CAM (chick chorioallantoic membrane) assay. From the generated compound library, compounds 16 and 19 proved to be potent and selective inhibitors of alpha IIb beta 3 (IC50 = 14 nM) whereas compound 11 showed excellent in vivo inhibition of angiogenesis (at 30 micrograms/embryo).

Allantois↗
PubMed1998

Detection of tumor angiogenesis in vivo by alphaVbeta3-targeted magnetic resonance imaging.

Angiogenesis, the formation of new blood vessels, is a requirement for malignant tumor growth and metastasis. In the absence of angiogenesis, local tumor expansion is suppressed at a few millimeters and cells lack routes for distant hematogenous spread. Clinical studies have demonstrated that the degree of angiogenesis is correlated with the malignant potential of several cancers, including breast cancer and malignant melanoma. Moreover, the expression of a specific angiogenesis marker, the endothelial integrin alphaVbeta3, has been shown to correlate with tumor grade. However, studies of tumor angiogenesis such as these have generally relied on invasive procedures, adequate tissue sampling and meticulous estimation of histologic microvessel density. In the present report, we describe a novel approach to detecting angiogenesis in vivo using magnetic resonance imaging (MRI) and a paramagnetic contrast agent targeted to endothelial alphaVbeta3 via the LM609 monoclonal antibody. This approach provided enhanced and detailed imaging of rabbit carcinomas by directly targeting paramagnetic agents to the angiogenic vasculature. In addition, angiogenic 'hot spots' not seen by standard MRI were detected. Our strategy for MR imaging of alphaVbeta3 thus represents a non-invasive means to assess the growth and malignant phenotype of tumors.

Animals↗
PubMed1998

Adenovirus endocytosis via alpha(v) integrins requires phosphoinositide-3-OH kinase.

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by alpha(v) integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas alpha(v) integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with alpha(v), integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.

Adenoviruses, Human↗
PubMed1998

Reduced intimal thickening following alpha(v)beta3 blockade is associated with smooth muscle cell apoptosis.

The adhesion integrin alpha(v)beta3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of alpha(v)beta3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of alpha(v)beta3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of alpha(v)beta3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of alpha(v)beta3. At both 2 and 4 wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of alpha(v)beta3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing alpha(v)beta3. These findings may explain the reduction in restenosis observed clinically following beta3 integrin blockade.

Animals↗