Integrin αvß3 Receptor Overexpressing on Tumor-Targeted Positive MRI-Guided Chemotherapy.

Multifunctional nanomaterials with targeted imaging and chemotherapy have high demand with great challenge. Herein, we rationally aimed to design multifunctional drug delivery systems by RGD-modified chitosan (CH)-coated nanoneedles (NDs) of gadolinium arsenate (RGD-CH-Gd-AsNDs). These NDs have multifunctionality for imaging and targeted therapy. NDs on intravenous administration demonstrated significant accumulation of As ions/species in tumor tissues, which was monitored by the change in T1-weighted magnetic resonance (MR) imaging. Moreover, NDs were well opsonized in cells with high specificity, subsequently inducing apoptosis to the HepG2 cells. Consequent to this, the in vivo results demonstrated biosafety, enhanced tumor targeting, and tumor regression in a subcutaneously transplanted xenograft model in nude mice. These RGD-CH-Gd-AsNDs have great potential, and we anticipate that they could serve as a novel platform for real-time T1-weighted MR diagnosis and chemotherapy.

Could Dissecting the Molecular Framework of ß-Lactam Integrin Ligands Enhance Selectivity?

By dissecting the structure of ß-lactam-based ligands, a new series of compounds was designed, synthesized, and evaluated toward integrins αvß3, α5ß1, and α4ß1. New selective ligands with antagonist or agonist activities of cell adhesion in the nanomolar range were obtained. The best agonist molecules induced significant adhesion of SK-MEL-24 cells and Saos-2 cells as a valuable model for osteoblast adhesion. These data could lead to the development of new agents to improve cellular osseointegration and bone regeneration. Molecular modeling studies on prototypic compounds and αvß3 or α5ß1 integrin supported the notion that ligand carboxylate fixing to the metal ion-dependent adhesion site in the ß-subunit can be sufficient for binding the receptors, while the aryl side chains play a role in determining the selectivity as well as agonism versus antagonism.

Systemic overexpression of matricellular protein CCN1 exacerbates obliterative bronchiolitis in mouse tracheal allografts.

Obliterative bronchiolitis (OB) involves airway epithelial detachment, fibroproliferation, and inflammation, resulting in chronic rejection and transplant failure. Cysteine-rich 61 (CCN1) is an integrin receptor antagonist with a context-dependent role in inflammatory and fibroproliferative processes. We used a mouse tracheal OB model to investigate the role of CCN1 in the development of lung allograft OB. C57Bl/6 mice received a systemic injection of CCN1-expressing adenoviral vectors 2 days prior to subcutaneous implantation of tracheal allografts from major MHC-mismatched BALB/c mice. We treated another group of tracheal allograft recipients with cyclic arginine-glycine-aspartic acid peptide to dissect the role of αvß3-integrin signaling in mediating CCN1 effects in tracheal allografts. Allografts were removed 4 weeks after transplantation and analyzed for luminal occlusion, inflammation, and vasculogenesis. CCN1 overexpression induced luminal occlusion (P < 0.05), fibroproliferation, and smooth muscle cell proliferation (P < 0.05). Selective activation of αvß3-integrin receptor failed to mimic the actions of CCN1, and blocking failed to inhibit the effects of CCN1 in tracheal allografts. In conclusion, CCN1 exacerbates tracheal OB by enhancing fibroproliferation via an αvß3-integrin-independent pathway. Further experiments are required to uncover its potentially harmful role in the development of OB after lung transplantation.

Targeting integrins αvß3 and α5ß1 with new ß-lactam derivatives.

The αvß3 and α5ß1 integrins are widely expressed in different cancer types and recognize the tripeptide Arg-Gly-Asp (RGD) motif present in several extracellular matrix proteins. We report here the design, synthesis and biological activity of some new ß-lactam derivatives specifically designed to target integrins. The new molecules contain the azetidinone as the only cyclic framework armed with carboxylic acid and amine terminals spaced from 9 to 14 atoms to switch on recognition by integrins. All tested molecules showed a concentration-dependent enhancement in fibronectin-mediated adhesion of K562 and SK-MEL-24 cells; in particular 1, expressed a higher affinity towards α5ß1 integrin (EC50 of 12 nM) and 2 was more selective for integrin αvß3 (EC50 of 11 nM).

Small molecule hormone or hormone-like ligands of integrin αVß3: implications for cancer cell behavior.

Integrins are heterodimeric structural components of the plasma membrane whose ligands include a large number of extracellular matrix (ECM) proteins. The ligands contain Arg-Gly-Asp (RGD) sequences that enable recognition of ECM proteins by as many as eight integrins, but other distinguishing features of the proteins permit the integrins to generate intracellular signals specific to the ECM molecules. Recently, integrin αvß3 has been shown to have a panel of previously unappreciated small molecule receptor sites for thyroid hormone and hormone analogues, for dihydrotestosterone, and for resveratrol, a polyphenol that has certain estrogen-like features. These binding sites are close to the RGD recognition site of αvß3. The thyroid hormone receptor site on the extracellular domain of αvß3 contains two domains with discrete functions in terms of intracellular protein trafficking and gene expression. Occupancy of the receptor by a deaminated thyroid hormone analogue, tetraiodothyroacetic acid (tetrac), prevents cell responses to agonist thyroid hormones (L-thyroxine; 3, 5, 3'-triiodo-L-thyronine) and modulates expression of a number of cancer cell survival pathway genes in an up- or downregulation pattern coherent to induction of cell death. The small molecule thyroid hormone receptor on the integrin also regulates activity of five vascular growth factor receptors and/or their ligands, providing control of angiogenesis via specific pharmacologic regulation of this thyroid hormone receptor. The resveratrol receptor induces programmed cancer cell death via p53, even when the latter has undergone specific mutations. There is also evidence for the presence of several receptors on integrin αvß3 for authentic steroids, including a dihydrotestosterone site that supports proliferation of breast cancer cells that lack nuclear androgen and estrogen receptors. The existence of these small molecule hormone receptors on an integrin with a remarkably complex functional profile defines novel pharmacologic options via individual small molecule receptor manipulation for control of cancer cell behavior. This refinement of up-down control at the level of discrete receptors is not a function of the use of αvß3 antibody or RGD peptides that occlude regions of the integrin.

Contribution of distinct platelet integrins to binding, unfolding, and assembly of fibronectin.

Fibronectin (FN) fibrillogenesis depends on the binding of FN to cellular receptors and subsequent unfolding of bound FN. Integrins αIIbß3, αvß3, and α5ß1 are known to assemble FN fibrils on platelets. In our study, we examined the contribution of these integrins to FN binding, unfolding, and assembly on platelets in suspension and adherent platelets in the presence or absence of agonists. Phorbol 12-myristate 13-acetate (PMA), but not adenosine diphosphate (ADP), induced binding of FN to platelets in suspension. In contrast, adherent platelets were able to deposit FN on their surfaces in the absence of agonists. ß3 integrins had a major impact on the interaction of FN on platelets. αvß3 showed a similar contribution to the binding of FN as αIIbß3 on PMA-stimulated platelets in suspension but had a lesser contribution to unfolding and deposition of FN on adherent platelets. α5ß1 also participated in the interaction of FN with platelets by mediating the unfolding and assembly of FN, but to a lesser extent than ß3 integrins. None of the distinct antibodies directed against one of the three integrins caused a complete inhibition of binding, unfolding, and assembly of FN by platelets. Thus, it is likely that αIIbß3, αvß3, and α5ß1 or another still unknown receptor can be substituted.

(68)Ga-labeled NOTA-RGD-BBN peptide for dual integrin and GRPR-targeted tumor imaging.

PURPOSE: Radiolabeled Arg-Gly-Asp (RGD) and bombesin (BBN) peptide analogs have been extensively investigated for the imaging of tumor integrin alpha(v)beta(3) and gastrin-releasing peptide receptor (GRPR) expression, respectively. Recently, we designed and synthesized a RGD-BBN heterodimeric peptide from c(RGDyK) and BBN(7-14) through a glutamate linker. The goal of this study was to investigate the dual receptor-targeting property and tumor diagnostic value of RGD-BBN heterodimeric peptide labeled with generator-eluted (68)Ga (t(1/2) 68 min, beta(+) 89% and EC 11%), (68)Ga-NOTA-RGD-BBN. METHODS: RGD-BBN heterodimer was conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) and labeled with (68)Ga. The dual receptor binding affinity was investigated by a radioligand competition binding assay. The in vitro and in vivo dual receptor targeting of (68)Ga-NOTA-RGD-BBN was evaluated and compared with that of (68)Ga-NOTA-RGD and (68)Ga-NOTA-BBN. RESULTS: NOTA-RGD-BBN had integrin alpha(v)beta(3) and GRPR binding affinities comparable to those of the monomeric RGD and BBN, respectively. The dual receptor targeting property of (68)Ga-NOTA-RGD-BBN was validated by blocking studies in a PC-3 tumor model. (68)Ga-NOTA-RGD-BBN showed higher tumor uptake than (68)Ga-NOTA-RGD and (68)Ga-NOTA-BBN. (68)Ga-NOTA-RGD-BBN can also image tumors with either integrin or GRPR expression. CONCLUSION: (68)Ga-NOTA-RGD-BBN exhibited dual receptor targeting properties both in vitro and in vivo. The favorable characterizations of (68)Ga-NOTA-RGD-BBN such as convenient synthesis, high specific activity, and high tumor uptake, warrant its further investigation for clinical cancer imaging.

Molecular basis of platelet activation by an alphaIIbbeta3-CHAMPS peptide.

BACKGROUND: A novel method, known as computed helical anti-membrane protein (CHAMP), for the design of peptides that bind with high affinity and selectivity to transmembrane helices was recently described and illustrated using peptides that bind alphaIIb- and alphav-integrin subunits, which induce selective activation of integrins alphaIIbbeta3 and alphavbeta3, respectively. OBJECTIVES: In the present study, we have investigated the ability of an alphaIIb-CHAMPS peptide (termed integrin-activatory-peptide or IAP) to stimulate protein tyrosine phosphorylation and aggregation in human and mouse platelets. METHODS: The ability of IAP to stimulate platelet aggregation and dense granule secretion was measured in washed preparations of human and mouse platelets. Samples were taken for measurement of tyrosine phosphorylation. RESULTS: IAP stimulates robust tyrosine phosphorylation of the tyrosine kinase Syk and the FcR gamma-chain, but only weak phosphorylation of PLCgamma2. Aggregation to low but not high concentrations of IAP is reduced in the presence of the Src kinase inhibitor, PP1, or by inhibitors of the two feedback agonists, ADP and thromboxane A(2) (TxA(2)) suggesting that activation is reinforced by Src kinase-driven release of ADP and TxA(2). Unexpectedly, aggregation by IAP is only partially inhibited in human and mouse platelets deficient in integrin alphaIIbbeta3. Further, IAP induces partial aggregation of formaldehyde-fixed platelets. CONCLUSIONS: The present study demonstrates that the alphaIIb-CHAMPS peptide induces platelet activation through integrin alphaIIbbeta3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR gamma-chain and Syk. The use of the alphaIIb-CHAMPS peptide to study integrin alphaIIbbeta3 function is compromised by non-integrin-mediated effects.

Rational design of highly active and selective ligands for the alpha5beta1 integrin receptor.

The inhibition of integrin function is a major challenge in medicinal chemistry. Potent ligands are currently in different stages of clinical trials for the antiangiogenic therapy of cancer and age-related macula degeneration (AMD). The subtype alpha5beta1 has recently been drawn into the focus of research because of its genuine role in angiogenesis. In our previous work we could demonstrate that the lack of structural information about the receptor could be overcome by a homology model based on the X-ray structure of the alphavbeta3 integrin subtype and the sequence similarities between both receptors. In this work, we describe the rational design and synthesis of high affinity alpha5beta1 binders, and the optimisation of selectivity against alphavbeta3 by means of extensive SAR studies and docking experiments. A first series of compounds based on the tyrosine scaffold resulted in affinities in the low and even subnanomolar range and selectivities of 400-fold against alphavbeta3. The insights about the structure-activity relationship gained from tyrosine-based ligands could be successfully transferred to ligands that bear an aza-glycine scaffold to yield alpha5beta1 ligands with affinities of approximately 1 nm and selectivities that exceed 10(4)-fold. The ligands have already been successfully employed as selective alpha5beta1 ligands in biological research and might serve as lead structures for antiangiogenic cancer therapy.

Integrin alphavbeta3, requirement for VEGFR2-mediated activation of SAPK2/p38 and for Hsp90-dependent phosphorylation of focal adhesion kinase in endothelial cells activated by VEGF.

Endothelial cell migration, a key process in angiogenesis, requires the coordinated integration of motogenic signals elicited by the adhesion of endothelial cells to extracellular matrices and by angiogenic cytokines such as the vascular endothelial growth factor (VEGF). In this study, we found that addition of VEGF to human umbilical vein endothelial cells cultivated on vitronectin triggers a synergistic interaction between the VEGF receptor VEGFR2 and the clustered integrin receptor alphavbeta3. The interaction between VEGFR2 and alphavbeta3 is required for full phosphorylation of VEGFR2 and to drive the activation of motogenic pathways involving focal adhesion kinase (FAK) and stress-activated protein kinase-2/p38 (SAPK2/p38). The signal emanating from the VEGFR2 and alphavbeta3 interaction and leading to SAPK2/p38 activation proceeds directly from VEGFR2. The chaperone Hsp90 is found in a complex that coprecipitates with inactivated VEGFR2, and the association is increased by VEGF and decreased by geldanamycin, a specific inhibitor of Hsp90-mediated events. Geldanamycin also impairs the phosphorylation of FAK that results from the interaction between VEGFR2 and alphavbeta3, and this is accompanied by an inhibition of the recruitment of vinculin to VEGFR2. We conclude that a necessary cross talk should occur between VEGFR2 and the integrin alphavbeta3, to transduce the VEGF signals to SAPK2/p38 and FAK and that Hsp90 is instrumental in the building up of focal adhesions by allowing the phosphorylation of FAK and the recruitment of vinculin to VEGFR2.

Arteriolar flow recruitment with vitronectin receptor stimulation linked to remote wall shear stress.

Our purpose was to investigate the hemodynamic mechanism of flow recruitment within arteriolar networks as stimulated by the vitronectin receptor. Since remote stimulation of the vitronectin receptor initiates a flow-mediated dilation, we tested whether the response could be mimicked by exogenous NO. Arteriolar diameter, red blood cell flux, and velocity were measured in terminal branch arterioles arising from a central feed (8-10 microm) arteriole (together previously defined as an arteriolar network) in the cheek pouch tissue of anesthetized hamsters (n = 29, pentobarbital 70 mg/kg). Terminal branch arterioles were stimulated (micropipette, 10 s) with 10 microg/ml LM609 (agonist, vitronectin receptor), 10(-4) M nitroprusside (SNP), or control. The same amount of flow was recruited into the network by applying LM609 or SNP to only one branch of the network; however, flow distribution differed. With LM609, all recruited flow went directly to the branch that was stimulated. With SNP, recruited flow was distributed evenly throughout the network, no matter where the stimulus was applied. Thus, flow-mediated dilation induced by vitronectin receptor stimulation initiates a robust flow recruitment response, directing flow to the stimulus flow path. In contrast, NO alone initiates flow recruitment to the entire network, along multiple parallel flow paths.