Development of a bifunctional chelating agent containing isothiocyanate residue for one step F-18 labeling of peptides and application for RGD labeling

We report herein a novel isothiocyanate active ligand for fluorine-18 labeling prepared by four step synthesis. It can be conjugated to a target molecule containing an amino functional group under weak basic conditions by way of thiourea bond formation. We explored the application of synthesized ligand by conjugating to well known α_vβ₃ integrin targeting peptide, c(RGDyK). The conjugated peptide showed good radiochemical yield and efficiency with an excellent radiochemical purity (97.1 ± 1.2%) in a short reaction time (10 min). Labeled peptide showed excellent in vitro and in vivo stability (>95%). α_vβ₃ integrin specific tumor uptake was observed both in biodistribution and small animal microPET studies on α_vβ₃-positive U87MG (human glioma cells) xenograft bearing mice. In general, successful application of synthesized ligand for labeling of RGD peptide could facilitate the possibility of using this ligand for labeling peptides containing an amino functional group.     

A novel concept of radiosynthesis of a 99mTc-labeled dimeric RGD peptide as a potential radiotracer for tumor imaging

Radiolabeled Arg-Gly-Asp (RGD) peptides are promising agents for non invasive imaging of α_vβ₃ expression in malignant tumors. The integrin α_vβ₃ binding affinity and consequent tumor uptake could be improved when a dimeric RGD peptide is used as the targeting moiety instead of a monomer. Towards this, a novel approach was envisaged to synthesize a ^99mTc labeled dimeric RGD derivative using a RGD monomer and [^99mTcN]⁺² intermediate. The dithiocarbamate derivative of cyclic RGD peptide G₃-c(RGDfK) (G₃ = Gly-Gly-Gly, f = Phe, K = Lys) was synthesized and radiolabeled with [^99mTcN]⁺² intermediate to form the ^99mTcN-[G₃-c(RGDfK)]₂ complex in high yield (～98%). Biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors showed good tumor uptake [4.61 ± 0.04% IA/g at 30 min post-injection] with fast clearance of the activity from non-target organs/tissue. Scintigraphic imaging studies showed visible accumulation of activity in the tumor with appreciable target to background ratio.     

Impact of bifunctional chelators on biological properties of <Superscript>111</Superscript>In-labeled cyclic peptide RGD dimers

The present study describes the synthesis and biological evaluation of ¹¹¹In(DOTA-3P-RGD₂) (DOTA = 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid; 3P-RGD₂ = PEG₄-E[PEG₄-c(RGDfK)]₂; PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), ¹¹¹In(DTPA-3P-RGD₂) (DTPA = diethylenetriaminepentaacetic acid) and ¹¹¹In(DTPA-Bn-3P-RGD₂) (DTPA-Bn = 2-(p-thioureidobenzyl)-diethylenetriaminepentaacetic acid) as potential radiotracers for imaging tumor integrin α_vβ₃ expression in athymic nude mice bearing U87MG glioma xenografts. The aim of the study is to assess the impact of the bifunctional chelator (BFC) (DOTA vs. DTPA or DTPA-Bn) on the biodistribution characteristics of the ¹¹¹In-labeled 3P-RGD₂. IC₅₀ values of DOTA-3P-RGD₂, DTPA-3P-RGD₂ and DTPA-Bn-3P-RGD₂ were determined to be 1.3 ± 0.2, 1.4 ± 0.3, 1.3 ± 0.3 nM, respectively, against ¹²⁵I-c(RGDyK) bound to U87MG human glioma cells. Radiotracers were prepared by reacting ¹¹¹InCl₃ with the RGD peptide conjugates in NH₄OAc buffer (100 mM, pH 5.5). For DOTA-3P-RGD₂, successful radiolabeling could be completed by heating the reaction mixture at 100°C for 15–20 min. For DTPA-3P-RGD₂ and DTPA-Bn-3P-RGD₂, the radiolabeling was almost instantaneous at room temperature. The specific activity was ~50 mCi/mg (or ~100 mCi/μmol) for ¹¹¹In(DOTA-3P-RGD₂) and ~200 mCi/mg (or ~400 mCi/μmol) for ¹¹¹In(DTPA-3P-RGD₂). The results from biodistribution studies showed that all the three radiotracers have high tumor uptake and excellent tumor-to-background (T/B) ratios up to 4-h postinjection. After that time point, both ¹¹¹In(DTPA-3P-RGD₂) and ¹¹¹In(DTPA-Bn-3P-RGD₂) showed a much faster tumor washout and poorer T/B ratios than ¹¹¹In(DOTA-3P-RGD₂). The tumor uptake of ¹¹¹In(DOTA-3P-RGD₂) is integrin α_vβ₃- and RGD-specific. ¹¹¹In(DOTA-3P-RGD₂) is metabolically stable while only ~25% of ¹¹¹In(DTPA-Bn-3P-RGD₂) remains intact in the feces during 2-h period. On the basis of results from this study, it was concluded that ¹¹¹In(DTPA-3P-RGD₂) can be an effective integrin α_vβ₃-targeted radiotracer if the high-specific activity is required. However, DOTA remains to be the BFC of choice for the development of therapeutic lanthanide radiotracers.     

Design and synthesis of novel dual-cyclic RGD peptides for αvβ3 integrin targeting

The specific binding of RGD cyclic peptide with integrin α_vβ₃ attracts great research interest for tumor-targeting drug delivery. Herein, we designed and synthesized a series of dual-ring RGD-peptide derivatives as a drug carrier for α_vβ₃ targeting. Three novel peptides showed excellent cell adhesion inhibition effect, in which, P3 exhibited 7-fold enhancement in IC₅₀ compared with cyclo(RGDfK). Drug-loaded cytotoxicity experiment and imaging experiment indicated that such dual-cyclic RGD peptides have good tumor targeting effects. This work provides a new strategy for the design of novel RGD peptides.     

Insights into binding modes of tumstatin peptide T7 with the active site of αvβ3 integrin

Through interaction with the active site of α_vβ₃ integrin, tumstatin T7 peptide inhibits both the angiogenesis and the proliferation of tumour cells. In this work, docking in conjunction with molecular dynamics simulation was used to explore the binding mode of T7 peptide and α_vβ₃ integrin. The binding mode analysis revealed that the residues Ser⁹⁰, Arg⁹¹, Asp⁹³ and Tyr⁹⁴ in T7 peptide, and (α)-Asp¹⁵⁰, (β)-Arg²¹⁴, (α)-Asp¹⁴⁸ (α)-Gln²¹⁴ and (α)-Glu¹²³ in the active site of α_vβ₃ integrin were most likely the key interaction sites. The hydroxyl of Tyr⁹⁴ coordinates α_vβ₃ via a Mn²⁺ ion, revealing that Mn²⁺ is also an important factor for the interaction. The insight into these key interaction sites not only suggests that the active site of α_vβ₃ integrin can bind to molecules through multiple binding mechanisms, but also provides some useful information for structure-based drug design.     

Exploring new near-infrared fluorescent disulfide-based cyclic RGD peptide analogs for potential integrin-targeted optical imaging

We synthesized disulfide-based cyclic RGD pentapeptides bearing a near-infrared fluorescent dye (cypate), represented by cypate-c(CRGDC) (1) for integrin-targeted optical imaging. These compounds were compared with the traditional lactam-based cyclic RGD counterpart, cypate-c(RGDfK) (2). Molecular modeling suggests that the binding affinity of 2 to integrin α_vβ₃ is an order of magnitude higher than that of 1. This was confirmed experimentally, which further showed that substitution of Gly with Pro, Val and Tyr in 1 remarkably hampered the α_vβ₃ binding. Interestingly, cell microscopy with A549 cells showed that 1 exhibited higher cellular staining than 2. These results indicate that factors other than receptor binding affinity to α_vβ₃ dimeric proteins mediate cellular uptake. Consequently, 1 and its analogs may serve as valuable molecular probes for investigating the selectivity and specificity of integrin targeting by optical imaging.     

Coexpression and characterization of the human large-conductance Ca2+-activated K+ channel α + β1 subunits in HEK293 cells

Large-conductance Ca²⁺-activated K⁺ channel is formed by a tetramer of the pore-forming α-subunit and distinct accessory β-subunits (β1–β4) which contribute to BK_Ca channel molecular diversity. Accumulative evidences indicate that not only α-subunit alone but also the α + β subunit complex and/or β-subunit might play an important role in modulating various physiological functions in most mammalian cells. To evaluate the detailed pharmacological and biophysical properties of α + β1 subunit complex or β1-subunit in BK_Ca channel, we established an expression system that reliably coexpress hSloα + β1 subunit complex in HEK293 cells. The coexpression of hSloα + β1 subunit complex was evaluated by western blotting and immunolocalization, and then the single-channel kinetics and pharmacological properties of expressed hSloα + β1 subunit complex were investigated by cell-attached and outside-out patches, respectively. The results in this study showed that the expressed hSloα + β1 subunit complex demonstrated to be fully functional for its typical single-channel traces, Ca²⁺-sensitivity, voltage-dependency, high conductance (151 ± 7 pS), and its pharmacological activation and inhibition.     

Engineered Cystine-Knot Peptides that Bind αvβ3 Integrin with Antibody-Like Affinities

The α_vβ₃ integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4-kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to α_vβ₃ integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a six amino acid loop of AgRP with a nine amino acid loop containing the Arg-Gly-Asp integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting to identify clones with high affinity to detergent-solubilized α_vβ₃ integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing α_vβ₃ integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown to bind specifically to α_vβ₃ integrins and had only minimal or no binding to α_vβ₅, α₅β₁, and α_iibβ₃ integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally occurring ligand for α_vβ₃ and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second-generation libraries by individually randomizing these loops in one of the high-affinity integrin-binding variants. Screening of these loop-randomized libraries against α_vβ₃ integrins resulted in peptides that retained high affinities for α_vβ₃ and had increased specificities for α_vβ₃ over α_iibβ₃ integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.     

αvβ5 Integrin is Localized at Focal Contacts by HT-1080 Fibrosarcoma Cells and Human Skin Fibroblasts Attached to Vitronectin

In this study we characterized α_vβ₅ integrin on HT-1080 fibrosarcoma cells. First, α_vβ₅ integrin was immunoprecipitated by ¹²⁵I-surface labeled HT-1080 cells using a polyclonal antibody specific for β₅ subunit (cytoplasmic domain). A heterodimer consisting of a β₅-chain running at 100 kD (reduced) and 90 kD (non-reduced) associated with an α-chain 145 kD (non-reduced) and 125 kD (reduced) was obtained by SDS-PAGE and autoradiography. By double-immunofluorescence labeling, we then investigated α_vβ₅ distribution on HT-1080 cells. Upon staining with anti-β₅ subunit antibody, α_vβ₅ was detected in focal contacts on cells attached to vitronectin (vn), co-localizing with vinculin at the end of actin filaments. Comparative analysis of α_vβ₅ and α_vβ₃ showed that both receptors can occupy the same focal contact, although on the same cell mostly they are clustered in independent focal contacts. Focal distribution of α_vβ₅ was also found on normal human fibroblasts attached to vn, suggesting that this is not a specific feature of HT-1080 cells. Finally, we investigated the role of α_vβ₅ and α_vβ₃ integrins in mediating HT-1080 cell adhesion to vn. Inhibition studies using antibodies with function-blocking activity to α_vβ₅ and α_vβ₃ suggest a primary role of α_vβ₅ to support cell adhesion, with a weak contribute of α_vβ₃. Their activity can be modulated by divalent cations. Our results provide the first evidence of focal distribution of α_vβ₅ integrin on cells attached to vn.     

αvβ3-Integrin-targeting lanthanide complex: synthesis and evaluation as a tumor-homing luminescent probe

The application of lanthanide complexes in the time-resolved fluorescence imaging of living cells has emerged in the last few decades, providing high-contrast images of cells through detection of the delayed emission. In the present study, we synthesized novel trivalent lanthanide complexes containing the cyclic peptide c(RGDfK) to visualize the α_vβ₃-integrin-expressing tumor cells. Conjugation of c(RGDfK) with the macrocyclic bipyridine ligand had little effect on the fluorescence properties of the complex, indicating that the coordinated lanthanide ion was well isolated from the peptide. Bright luminescence images of α_vβ₃-integrin-expressing U87-MG cells were successfully obtained by employing the probes.     

Cyclic RGD peptide-labeled upconversion nanophosphors for tumor cell-targeted imaging

One of the great challenges of oncology is to improve methods for early tumor detection. Thus tumor cell-targeted optical imaging has been intensively studied. Bioimaging with upconversion (UC) phosphors (UCPs) is of considerable interest due to a variety of possible applications taking advantage of infrared-to-visible luminescence. Here we report for the first time tumor cell-targeted UC imaging using UCPs modified with cyclic RGD peptide (RGD-Y₂O₃). Cyclic RGD peptide binds specifically to integrin α_vβ₃ which is highly expressed in a tumor cell surface of certain cancer types but not in normal tissues. Since UC emission from RGD-Y₂O₃ was observed for U87MG cancer cell (high integrin α_vβ₃ expression), but not for MCF-7 cancer cell (low integrin α_vβ₃ expression), this UC imaging is considered to be integrin α_vβ₃ specific. The non-invasive imaging of integrin α_vβ₃ expression using UCP-based probes will have great potential in cancer imaging in general in living subjects.     

A novel radiofluorinated agouti-related protein for tumor angiogenesis imaging

A novel protein scaffold based on the cystine knot domain of the agouti-related protein (AgRP) has been used to engineer mutants that can bind to the α_vβ₃ integrin receptor with high affinity and specificity. In the current study, an ¹⁸F-labeled AgRP mutant (7C) was prepared and evaluated as a positron emission tomography (PET) probe for imaging tumor angiogenesis. AgRP-7C was synthesized by solid phase peptide synthesis and site-specifically conjugated with 4-nitrophenyl 2-^18/19F-fluoropropionate (^18/19F-NFP) to produce the fluorinated peptide, ^18/19F-FP-AgRP-7C. Competition binding assays were used to measure the relative affinities of AgRP-7C and ¹⁹F-FP-AgRP-7C to human glioblastoma U87MG cells that overexpress α_vβ₃ integrin. In addition, biodistribution, metabolic stability, and small animal PET imaging studies were conducted with ¹⁸F-FP-AgRP-7C using U87MG tumor-bearing mice. Both AgRP-7C and ¹⁹F-FP-AgRP-7C specifically competed with ¹²⁵I-echistatin for binding to U87MG cells with half maximal inhibitory concentration (IC₅₀) values of 9.40 and 8.37 nM, respectively. Non-invasive small animal PET imaging revealed that ¹⁸F-FP-AgRP-7C exhibited rapid and good tumor uptake (3.24 percentage injected dose per gram [% ID/g] at 0.5 h post injection [p.i.]). The probe was rapidly cleared from the blood and from most organs, resulting in excellent tumor-to-normal tissue contrasts. Tumor uptake and rapid clearance were further confirmed with biodistribution studies. Furthermore, co-injection of ¹⁸F-FP-AgRP-7C with a large molar excess of blocking peptide c(RGDyK) significantly inhibited tumor uptake in U87MG xenograft models, demonstrating the integrin-targeting specificity of the probe. Metabolite assays showed that the probe had high stability, making it suitable for in vivo applications. ¹⁸F-FP-AgRP-7C exhibits promising in vivo properties such as rapid tumor targeting, good tumor uptake, and excellent tumor-to-normal tissue ratios, and warrants further investigation as a novel PET probe for imaging tumor angiogenesis.     

Synthesis and evaluation of cyclic RGD-boron cluster conjugates to develop tumor-selective boron carriers for boron neutron capture therapy

Boron-containing agents play a key role in successful boron neutron capture therapy (BNCT). Icosahedral boron cluster-Arg-Gly-Asp (RGD) peptide conjugates were designed, synthesized, and evaluated for the biodistribution to develop tumor-selective boron carriers. Integrin αvβ3 is an attractive target for anti-tumor drug delivery because of its specific expression in proliferating endothelial and tumor cells of various origins. We, therefore, selected a c(RGDfK) moiety recognizing αvβ3 as an active tumor-targeting device to conjugate with icosahedral boron-10 clusters, disodium mercaptododecaborate (BSH) or o-carborane as a thermal neutron-sensitizing unit. Preparation of o-carborane derivatives involved microwave irradiation, and resulted in high yields in a short time. An in vitro cell adhesion assay on αvβ3-positive U87MG and SCCVII cells demonstrated the high binding affinity of conjugates to integrin αvβ3 (IC(50)=0.19-2.66 μM). Biodistribution experiments using SCCVII-bearing mice indicated that GPU-201 showed comparable tumor uptake and a significantly longer retention in tumors compared with BSH. These results suggest that GPU-201 is a promising candidate for use in BNCT.     

Vitronectin decreases microvascular endothelial cell apoptosis

Angiogenesis after tissue injury occurs in a matrix environment consisting of fibrin, fibronectin, and vitronectin as the major extracellular matrix (ECM) constituents. ECM-integrin interactions is critical for angiogenesis and failure to bind a ligand to certain integrin receptors (α_vβ₃ or α_vβ₅) inhibits angiogenesis. The ligand that binds to α_vβ₃ or α_vβ₅ integrin receptors during microvascular angiogenesis has not been identified. Our hypothesis is that provisional matrix molecules provide the environmental context cues to microvascular endothelial cells and promote angiogenesis by decreased programmed cell death. Using cultured human microvascular endothelial cells, we show that vitronectin, in comparison to growth on alternative provisional matrix molecules (fibronectin, fibrinogen plus thrombin), collagen I, and basement membrane molecules (collagen IV), significantly reduces microvascular endothelial cell death in vitro. This reduction was observed using morphologic criteria, TdT-mediated dUTP nick end labeling (TUNEL) assay, histone release into the cytoplasm, and thymidine release into the supernatant. Though our data confirm that vitronectin may bind to more than one integrin receptor to reduce MEC apoptosis, binding to the α_v component appears to be the critical integrin subcomponent for reducing apoptosis. J. Cell. Physiol. 175:149–155, 1998. © 1998 Wiley-Liss, Inc.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Synthesis and in vitro evaluation of a novel radioligand for αvβ3 integrin receptor imaging: [18F]FPPA-c(RGDfK)

The development of RGD-based antagonist of αvβ3 integrin receptor has enhanced the interest in PET probes to image this receptor for the early detection of cancer, to monitor the disease progression and the response to therapy. In this work, a novel prosthetic group (N-(4-fluorophenyl)pent-4-ynamide or FPPA) for the ¹⁸F-labeling of an αvβ3 selective RGD-peptide was successfully prepared. [¹⁸F]FPPA was obtained in three steps with a radiochemical yield of 44% (decay corrected). Conjugation to c(RGDfK(N₃)) by the Cu(II) catalyzed Huisgen azido alkyne cycloaddition provided the [¹⁸F]FPPA-c(RGDfK) with a radiochemical yield of 29% (decay corrected), in an overall synthesis time of 140 min.     

Localization of Urokinase Type Plasminogen Activator to Focal Adhesions Requires Ligation of Vitronectin Integrin Receptors

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the α_vβT₅ integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the α_vβT₅ integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either α_vβT₅ or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the βT₁ integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of α_vβT₃, α_vβT₅, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the α_vβT₃ or α_vβT₅ integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell-matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.     

The effects of estrogen,its antagonist ICI 182, 780, and interferon-tau on the expression of estrogen receptors and integrin alphaV beta 3 on cycle day 16 in bovine endometrium

We have shown previously that downregulation of intercaruncular stromal integrin α_vβ₃ in bovine endometrium on day 16 of the estrous cycle coincided with the antibody recognition of estrogen receptors (ER) in the luminal epithelium. In pregnancy, these changes were not observed. Our hypothesis was that on day 16 of the estrous cycle, estrogen from the dominant follicle causes a reduction in integrin α_vβ₃ and affects ERα in the luminal epithelium. The pregnancy recognition protein, interferon-τ (IFN-τ), may prevent downregulation of integrin α_vβ₃ and suppress ERα expression in the luminal epithelium. On days 14 to 16, heifers received uterine infusions of the anti-estrogen ICI 182, 780, estradiol 17β, IFN-τ or the saline control. On day 16, reproductive tracts were collected for analysis of integrin α_vβ₃ and ERα. Estrogen receptor α immunoreactivity was largely restricted to the luminal epithelium in control animals. Using anti-ERα recognizing the amino terminus, estrogen-treated animals showed reactivity in the stroma, shallow and deep glands and myometrium as is typical of estrus, whereas ICI 182, 870 treated heifers showed little or no reactivity. In contrast, carboxyl terminus-directed antibodies showed a widespread distribution of ERα with reactivity detected in the uterine epithelium, stroma and myometrium of both estrogen and ICI 182, 780 treated animals. Heifers treated with IFN-τ had low ERα reactivity overall. Control and IFN-τ treated heifers had lower intercaruncular stromal expression of integrin α_vβ₃ in comparison to estrogen and ICI 182, 780 treatments. Overall, the results suggest that on day 16 of the estrous cycle, estrogen effects on integrin α_vβ₃ are indirect and do not directly involve ERα in the luminal epithelium. During pregnancy, interferon-tau may block ERα in the luminal epithelium but likely does not rescue integrin α_vβ₃ expression.     

Characterization of adhesion properties of the cardiomyocyte integrins and extracellular matrix proteins using atomic force microscopy

Integrins are transmembrane adhesion receptors that play important roles in the cardiovascular system by interacting with the extracellular matrix (ECM). However, direct quantitative measurements of the adhesion properties of the integrins on cardiomyocyte (CM) and their ECM ligands are lacking. In this study, we used atomic force microscopy (AFM) to quantify the adhesion force (peak force and mean force) and binding probability between CM integrins and three main heart tissue ECM proteins, ie, collagen (CN), fibronectin (FN), and laminin (LN). Functionalizing the AFM probes with ECM proteins, we found that the peak force (mean force) was 61.69 ± 5.5 pN (76.54 ± 4.0 pN), 39.26 ± 4.4 pN (59.84 ± 3.6 pN), and 108.31 ± 4.2 pN (129.63 ± 6.0 pN), respectively, for the bond of CN‐integrin, FN‐integrin, and LN‐integrin. The binding specificity between CM integrins and ECM proteins was verified by using monoclonal antibodies, where α₁₀‐ and α₁₁‐integrin bind to CN, α₃‐ and α₅‐integrin bind to FN, and α₃‐ and α₇‐integrin bind to LN. Furthermore, adhesion properties of CM integrins under physiologically high concentrations of extracellular Ca²⁺ and Mg²⁺ were tested. Additional Ca²⁺ reduced the adhesion mean force to 68.81 ± 4.0 pN, 49.84 ± 3.3 pN, and 119.21 ± 5.8 pN and binding probability to 0.31, 0.34, 0.40 for CN, FN, and LN, respectively, whereas Mg²⁺ caused very minor changes to adhesion properties of CM integrins. Thus, adhesion properties between adult murine CM integrins and its main ECM proteins were characterized, paving the way for an improved understanding of CM mechanobiology.     

Morpholine-based RGD-cyclopentapeptides as αvβ3/αvβ5 integrin ligands: Role of configuration towards receptor binding affinity

Two c[RGDfX] cyclopeptides, having either l- or d-morpholine-3-COOH (Mor) as the X amino acid were developed as ligands for α_vβ₃/α_vβ₅ integrins. Biological assays showed only d-Mor-containing cyclopentapeptide capable to bind α_vβ₃ integrin with a low nanomolar affinity according to a two-site model, thus revealing a connection between the configuration of Mor and the preferred binding to α_vβ₃ integrin. Conformational analysis showed different structural preferences for the two peptides induced by the two enantiomeric cyclic amino acids, suggesting a role of the stereochemistry of Mor on the overall peptide conformation and on the presentation of the pharmacophoric Arg and Asp side chains.