Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies.     

Impaired cross-activation of β3 integrin and VEGFR-2 on endothelial progenitor cells with aging decreases angiogenesis in response to hypoxia

Background

The mechanism by which vascular regeneration declines with aging is not fully understood. An interaction between integrin and vascular endothelial growth factor receptor-2 (VEGFR-2) plays a substantial role in angiogenesis. Here, we investigated whether aging impairs this interaction in endothelial progenitor cells (EPCs) under hypoxia.

Methods and results

Aging reduced the blood flow and vessel density in ischemic muscles in mice. Levels of phosphorylated Src (p-Src), p-β₃, and p-VEGFR-2 in acute ischemia were reduced in the muscles of aged mice compared to young mice. The hypoxia-inducible factor (HIF)-1α stabilizer deferoxamine improved the age-related impairment of angiogenesis, but this effect was diminished by LY290004, an inhibitor of phosphatidylinositol 3-kinase. Deferoxamine improved the reduction in chronic ischemia-induced β₃-integrin and VEGFR-2 phosphorylation in the muscles of aged mice; this effect was also diminished by LY290004. In EPCs, we identified the molecular requirements for VEGF-mediated β₃-integrin and VEGFR-2 cross-activation in vitronectin-induced cell adhesion under acute hypoxia. We demonstrated that c-Src controlled the adhesion- and VEGF-induced β₃ tyrosine phosphorylation in hypoxia. Aging enhanced the hypoxia-induced EPC apoptosis and impaired several c-Src-related VEGF-induced receptor events, including β₃ tyrosine activation, ligand binding, cell adhesion, and tubulogenesis in cultured EPCs of animals and those of humans.

Conclusions

These data suggest that the aging-related decline in angiogenic action in response to ischemia is mediated by the impairment of cross-activation between β₃ and VEGFR-2 in EPCs, which is partially associated with decreased HIF-1α stability.     

Calpain regulation of integrin αIIb β3 signaling in human platelets

Efficient platelet adhesion and aggregation at sites of vascular injury requires the synergistic contribution of multiple adhesion receptors. The initial adhesion of platelets to subendothelial matrix proteins involves GPIb/ V/IX and one or more platelet integrins, including integrin α_IIb β₃ , α₂ β₁ , α₅ β₁ and possibly α₆ β₁. In contrast, platelet-platelet adhesion (platelet cohesion or aggregation) is mediated exclusively by GPIb/V/IX and integrin α_IIb β₃ . Integrin α_IIb β₃ is a remarkable receptor that not only stabilizes platelet-vessel wall and platelet-platelet adhesion contacts, but also transduces signals necessary for a range of other functional responses. These signals are linked to cytoskeletal reorganization and platelet spreading, membrane vesiculation and fibrin clot formation, and tension development on a fibrin clot leading to clot retraction. This diverse functional role of integrin α_IIb β₃ is reflected by its ability to induce the activation of a broad range of signaling enzymes that are involved in membrane phospholipid metabolism, protein phosphorylation, calcium mobilization and activation of small GTPases. An important calcium-dependent signaling enzyme involved in integrin α_IIb β₃ outside-in signaling is the thiol protease, calpain. This enzyme proteolyses a number of key structural and signaling proteins involved in cytoskeletal remodeling and platelet activation. These proteolytic events appear to play a potentially important role in modulating the adhesive and signaling function of integrin α_IIb β₃ .     

Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin αvβ₃

Vascular endothelial growth factor (VEGF) acting through VEGF receptor 2 (VEGFR2) on endothelial cells (ECs) is a key regulator of angiogenesis, a process essential for wound healing and tumor metastasis. Rap1a and Rap1b, 2 highly homologous small G proteins, are both required for angiogenesis in vivo and for normal EC responses to VEGF. Here we sought to determine the mechanism through which Rap1 promotes VEGF-mediated angiogenesis. Using lineage-restricted Rap1-knockout mice we show that Rap1-deficiency in endothelium leads to defective angiogenesis in vivo, in a dose-dependent manner. Using ECs obtained from Rap1-deficient mice we demonstrate that Rap1b promotes VEGF-VEGFR2 kinase activation and regulates integrin activation. Importantly, the Rap1b-dependent VEGF-VEGFR2 activation is in part mediated via integrin α(v)β(3). Furthermore, in an in vivo model of zebrafish angiogenesis, we demonstrate that Rap1b is essential for the sprouting of intersomitic vessels, a process known to be dependent on VEGF signaling. Using 2 distinct pharmacologic VEGFR2 inhibitors we show that Rap1b and VEGFR2 act additively to control angiogenesis in vivo. We conclude that Rap1b promotes VEGF-mediated angiogenesis by promoting VEGFR2 activation in ECs via integrin α(v)β(3). These results provide a novel insight into the role of Rap1 in VEGF signaling in ECs.     

Elevated expression of β1 and β2 integrins,intercellular adhesion molecule 1, and endothelial leukocyte adhesion molecule 1 in the skin of patients with systemic sclerosis of recent onset

Objective. To investigate the possible role of integrins and cell adhesion molecules in the pathogenesis of the mononuclear cell infiltration and fibrosis of skin that occurs in systemic sclerosis (SSc). Methods. The presence and topographic distribution of β₁, β₂, and β₄ integrins, as well as of endothelial leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1), was examined immunohistochemically in affected skin from 8 patients with rapidly progressive SSc of recent onset. The expression of the β₁ integrin gene was also investigated by in situ hybridization with a human sequence-specific complementary DNA. Results. The presence of β₁ integrin epitopes and the corresponding messenger RNA within inflammatory cells surrounding small vessels was demonstrated in SSc skin but not in normal skin. Lymphocytes positive for β₂ integrin were also found only in SSc skin, and they appeared in close proximity to small blood vessels and collagen bundles. Immunostaining for β₄ integrin epitopes revealed no differences between normal and SSc skin. ELAM-1 and ICAM-1 monoclonal antibodies, which identify epitopes indicative of endothelial cell activation, stained endothelial cells in SSc skin but not normal skin. Conclusion. These observations suggest that the complex interactions of β₁ and β₂ integrins, as well as ELAM-1 and ICAM-1, may be intimately involved in the pathogenesis of SSc, perhaps by mediating the homing and targeting of pathogenetic lymphocytes to the affected tissues.     

Nafamostat mesilate can prevent adhesion, invasion and peritoneal dissemination of pancreatic cancer thorough nuclear factor kappa-B inhibition

Background

Constitutive activation of nuclear factor kappa-B (NF-??B) contributes to the aggressive behavior of pancreatic cancer. Over-expression of downstream target genes of NF-??B such as intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) leads to the promotion of cell adhesion, angiogenesis, invasion and metastasis. We previously reported that nafamostat mesilate, a synthetic serine protease inhibitor, blocks NF-??B activation in pancreatic cancer. We hypothesized that nafamostat mesilate may inhibit cell adhesion, angiogenesis, invasion and metastases in peritoneal dissemination of pancreatic cancer.

Methods

In vitro, we assessed inhibition of NF-??B, phosphorylated I??B??, ICAM-1, VEGF and MMP-9 activity by nafamostat mesilate using human pancreatic cancer cell lines (AsPC-1, BxPC-3 and PANC-1). Changes in adhesion and invasion abilities of cancer cells were then evaluated by nafamostat mesilate treatment. In vivo, the efficacy of nafamostat mesilate treatment was assessed using peritoneal dissemination of pancreatic cancer in mice.

Results

In vitro, nafamostat mesilate inhibited activities of NF-??B, phosphorylated I??B??, ICAM-1, VEGF and MMP-9. Moreover, nafamostat mesilate not only inhibited cell adhesion and invasion but also increased the sensitivity of anoikis. In vivo, tumor growth using AsPC-1 cells of the treatment group was significantly slower, and survival rate was significantly better, than those in control group (p?Conclusion Nafamostat mesilate reduced peritoneal metastasis and prolonged survival of pancreatic cancer-bearing mice.     

Nitric Oxide Induces Angiogenesis and Upregulates αvβ3 Integrin Expression on Endothelial Cells

Nitric oxide (NO) has been implicated as a mediator of angiogenesis. However, its precise role in angiogenesis and its mechanism of action have not been established. We performed in vivo and in vitro angiogenesis assays using NO donor S-nitroso-N-acetylpenicillamine (SNAP) and NO synthase inhibitor N-iminoethyl- -ornithine (L-NIO). SNAP significantly increased and L-NIO significantly suppressed capillary ingrowth into subcutaneously implanted Matrigel plugs in mice. For the in vitro angiogenesis assay, human umbilical vein endothelial cells (HUVECs) (4 × 10⁴ cells/well) were treated with placebo, SNAP (100 μM), or L-NIO (100 μM) and cultured on Matrigel for 18 h. The typical capillary networks formed on Matrigel by HUVECs as a result of cell migration and differentiation were quantified by computer-assisted image analysis as a measure of angiogenesis. Treatment of HUVECs with SNAP significantly increased the capillary network area compared with control, 8701 ± 693 vs 6258 ± 622 area units (P < 0.05), whereas L-NIO significantly decreased the capillary area (4540 ± 342, P < 0.05). Furthermore, we have shown with a blocking monoclonal antibody that formation of capillary networks on Matrigel is mediated by the functional expression of the α_vβ₃ integrin, which plays a role in facilitating endothelial cell adhesion to basement membrane matrix and endothelial cell migration. After an 18-h culture, flow cytometry revealed that SNAP significantly upregulated and L-NIO significantly downregulated in a concentration-dependent manner α_vβ₃ integrin expression on endothelial cells. In conclusion, NO induces angiogenesis in vivo and in vitro by promoting endothelial cell migration and differentiation into capillaries. One possible mechanism might involve the upregulation of α_vβ₃ integrin on endothelial cells, a critical mediator of cell–matrix adhesion and migration.     

Structure of an integrin αIIbβ3 transmembrane-cytoplasmic heterocomplex provides insight into integrin activation

Heterodimeric integrin adhesion receptors regulate diverse biological processes including angiogenesis, thrombosis and wound healing. The transmembrane-cytoplasmic domains (TMCDs) of integrins play a critical role in controlling activation of these receptors via an inside-out signaling mechanism, but the precise structural basis remains elusive. Here, we present the solution structure of integrin αIIbβ3 TMCD heterodimer, which reveals a right-handed coiled-coil conformation with 2 helices intertwined throughout the transmembrane region. The helices extend into the cytoplasm and form a clasp that differs significantly from a recently published αIIbβ3 TMCD structure. We show that while a point mutation in the clasp interface modestly activates αIIbβ3, additional mutations in the transmembrane interface have a synergistic effect, leading to extensive integrin activation. Detailed analyses and structural comparison with previous studies suggest that extensive integrin activation is a highly concerted conformational transition process, which involves transmembrane coiled-coil unwinding that is triggered by the membrane-mediated alteration and disengagement of the membrane-proximal clasp. Our results provide atomic insight into a type I transmembrane receptor heterocomplex and the mechanism of integrin inside-out transmembrane signaling.     

Synergistic effect of peptide inhibitors derived from the extracellular and intracellular domain of αIIb subunit of integrin αIIbβ3 on platelet activation and aggregation

α_IIbβ₃, the major platelet integrin, plays a central role in hemostasis and thrombosis. Upon platelet activation, conformation of α_IIbβ₃ changes and allows fibrinogen binding and, subsequently, platelet aggregation. It was previously shown that a lipid-modified platelet permeable peptide, which corresponds to the intracellular acidic membrane distal sequence ¹⁰⁰⁰LEEDDEEGE¹⁰⁰⁸ of α_IIb (pal-K-LEEDDEEGE or pal-K-1000-1008), inhibits thrombin-induced human platelet aggregation, by inhibiting talin association with the integrin. YMESRADR, a peptide corresponding to the extracellular sequence 313–320 of α_IIb, is also a potent platelet aggregation inhibitor by mimicking the effect of a clasp between the head domains of α_IIb and β₃. The aim of the present study was to investigate the synergistic effect of the intra- and extracellular- peptide inhibitors on platelet aggregation, as well as on the phosphorylation of two signaling proteins, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Platelet preincubation with Pal-K-LEEDDEGE followed by YMESRADR showed a synergistic inhibitory activity on platelet aggregation. Platelet incubation with threshold inhibitory concentrations of both peptides provoked almost the total inhibition of aggregation, PAC-1 binding, and fibrinogen binding, but not P-selectin exposure on activated platelets’ surface. Like RGDS peptide, this mixture inhibits FAK phosphorylation whose phosphorylation is well known to be consecutive to the aggregation (postoccupancy events). However, in contrast to RGDS peptide that enhances ERK phosphorylation and activation, the mixture of threshold inhibitory concentrations of Pal-K-LEEDDEEGE and YMESRADR inhibits ERK phosphorylation. We suggest that the use of the intracellular in combination with the extracellular peptide inhibitor, acting with a non-RGD-like mechanism, may provide an alternative way to antagonize integrin α_IIbβ₃ activation.     

Integrin β1-focal adhesion kinase signaling directs the proliferation of metastatic cancer cells disseminated in the lungs

The development of metastases is an extended and inefficient process involving multiple steps. The last of these involves the growth of micrometastases into macroscopic tumors. We show here that intravenously injected, nonmetastatic cancer cells cease proliferating after extravasating into the parenchyma of the lungs; this response is attributable to the cells inability to trigger adhesion-related signaling events when they are scattered sparsely within the extracellular matrix (ECM) of the parenchyma. We recapitulate this situation by culturing these nonmetastatic cells at low seeding density in ECM-derived gels in vitro, in which they undergo cell-cycle arrest resulting, in part, from insufficient activation of focal adhesion kinase (FAK). Metastatic cancer cells, in contrast, show sufficient FAK activation to enable their proliferation within ECM gels in vitro and continue cell-cycle progression within the lung parenchyma in vivo. Activation of FAK in these metastatic cells depends on expression of the β₁ subunit of integrins, and proliferation of these cells after extravasation in the lungs is diminished by knocking down the expression of either FAK or integrin β₁. These results demonstrate the critical role of integrin β₁-FAK signaling axis in controlling the initial proliferation of micrometastatic cancer cells disseminated in the lungs.     

Role of Rap1 in promoting sickle red blood cell adhesion to laminin via BCAM/LU

Vaso-occlusion is a hallmark of sickle cell disease. Agonist-induced activation of sickle red blood cells (SS RBCs) promotes their adhesion to vascular proteins, potentially contributing to vasoocclusion. Previously, we described a cyclic adenosine monophosphate (cAMP)-dependent increase in SS RBC adhesion to laminin. Here, we investigated whether Rap1, a small guanosine triphosphatase (GTPase) known to promote integrin-mediated adhesion in other cells, was involved in this signaling pathway. We found that agonists known to induce cAMP signaling promoted the GTP-bound, active state of Rap1 in SS RBCs. The cAMP-dependent exchange factor Epac (exchange protein directly activated by cAMP) is a likely upstream activator of Rap1, since Epac is present in these cells and the Epac-specific cAMP analog 8CPT-2-Me (8-(4-cholorophenylthio)-2'-O-methyl-cAMP) activated Rap1 and promoted SS RBC adhesion to laminin. This 8CPT-2-Me-stimulated adhesion was integrin independent, since it was insensitive to RGD peptide or antibodies against the only known integrin on SS RBCs, alpha4beta1. However, this adhesion was completely inhibited by either a soluble version of basal cell adhesion molecule/Lutheran (BCAM/LU) or a BCAM/LU adhesion-blocking anti-body. Surprisingly, 8CPT-2-Me-activated Rap1 did not promote SS RBC adhesion to a known alpha4beta1 ligand, vascular cell adhesion molecule 1 (VCAM-1). These results demonstrate that Epac-induced Rap1 activation in SS RBCs promotes BCAM/LU-mediated adhesion to laminin. Thus, Epac-mediated Rap1 activation may represent an important signaling pathway for promoting SS RBC adhesion.     

Laminin stimulates spreading of platelets through integrin alpha6beta1-dependent activation of GPVI

The extracellular matrix protein, laminin, supports platelet adhesion through binding to integrin alpha6beta1 In the present study, we demonstrate that human laminin, purified from placenta, also stimulates formation of filopodia and lamellipodia in human and mouse platelets through a pathway that is dependent on alpha6beta1 and the collagen receptor GPVI. The integrin alpha6beta1 is essential for adhesion to laminin, as demonstrated using an alpha6-blocking antibody, whereas GPVI is dispensable for this response, as shown using "knockout" mouse platelets. On the other hand, lamellipodia formation on laminin is completely inhibited in the absence of GPVI, although filopodia formation remains and is presumably mediated via alpha6beta1 Lamellipodia and filopodia formation are inhibited in Syk-deficient platelets, demonstrating a key role for the kinase in signaling downstream of GPVI and integrin alpha6beta1 GPVI was confirmed as a receptor for laminin using surface plasmon resonance spectroscopy and by demonstration of lamellipodia formation on laminin in the presence of collagenase. These results identify GPVI as a novel receptor for laminin and support a model in which integrin alpha6beta1 brings laminin to GPVI, which in turn mediates lamellipodia formation. We speculate that laminin contributes to platelet spreading in vivo through a direct interaction with GPVI.     

Analysis of angiogenesis induced by local IGF-1 expression after myocardial infarction using microSPECT-CT imaging

Insulin-like growth factor-1 (IGF-1) has been found to exert favorable effects on angiogenesis in prior animal studies. This study explored the long-term effect of IGF-1 on angiogenesis using microSPECT-CT in infarcted rat hearts after delivering human IGF-1 gene by adeno-associated virus (AAV). Myocardial infarction (MI) was induced in Sprague-Dawley rats by ligation of the proximal anterior coronary artery and a total of 10¹¹ AAV-CMV-lacZ (control) or IGF-1 vectors were injected around the peri-infarct area. IGF-1 expression by AAV stably transduced heart muscle for up to 16 weeks post-MI and immunohistochemistry revealed a remarkable increase in capillary density. A ^99mTc-labeled RGD peptide (NC100692, GE Healthcare) was used to assess temporal and regional α_v integrin activation. Rats were injected with NC100692 followed by ²⁰¹Tl chloride and in vivo microSPECT-CT imaging was performed. After imaging, hearts were excised and cut for quantitative gamma-well counting (GWC). NC100692 retention was significantly increased in hypoperfused regions of both lacZ and IGF-1 rats at 4 and 16 weeks post-MI. Significantly higher activation of α_v integrin was observed in IGF-1 rats at 4 weeks after treatment compared with control group, although the activation was lower in the IGF-1 group at 16 weeks. Local IGF-1 gene delivery by AAV can render a sustained transduction and improve cardiac function post-MI. IGF-1 expression contributes to enhanced α_v integrin activation which is linked to angiogenesis. MicroSPECT-CT imaging with ^99mTc-NC100692 and quantitative GWC successfully assessed differences in α_v integrin activation between IGF-1-treated and control animals post-MI.     

Activation signal transduction by β1 integrin in T cells from patients with systemic lupus erythematosus

Objective

Beta 1 integrin is a representative adhesion molecule for cell–cell and cell–extracellular matrix interactions, and it provides costimulatory signals to T cells. However, the relevance of β1 integrin to T cell activation in systemic lupus erythematosus (SLE) remains unclear. We undertook this study to perform a quantitative and functional analysis of β1 integrin–mediated signaling to T cells in patients with SLE.

Methods

Expression of cell surface molecules was assessed by flow cytometric analysis. Engagement of β1 integrins was performed by crosslinking using a specific monoclonal antibody. To assess tyrosine kinases in β1 integrin–mediated signaling, the cells were transfected with a wild‐type (WT) focal adhesion kinase (FAK), a dominant‐negative truncation of the FAK, or a WT PTEN expression plasmid via nucleofection.

Results

Beta 1 integrin expression was significantly up‐regulated on peripheral blood T cells from patients with active SLE, particularly those with the complication of World Health Organization class IV nephritis, whereas CD28 was significantly decreased in patients with active SLE compared with normal individuals. Beta 1 integrin expression closely correlated with serum hypocomplementemia. Engagement of β1 integrin on T cells from patients with active SLE, but not on those from normal individuals, induced cell proliferation as well as CD40L expression on T cells. Up‐regulation of CD40L expression and T cell proliferation, induced by β1 integrin stimulation, were completely inhibited by transfection of the dominant‐negative truncations of FAK or WT PTEN.

Conclusion

These results suggest that engagement of β1 integrins on SLE T cells could induce FAK‐mediated signaling and subsequent CD40L expression and proliferation. Thus, the β1 integrin signaling cascade might serve to enhance autoreactive T cell activation.

     

Complex interactions between the laminin alpha 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo

The alpha4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins alphavbeta3, alpha3beta1, and alpha6beta1. An antibody against the G domain (residues 919-1207; G(919-1207)) of the alpha4 laminin subunit inhibits angiogenesis in a mouse-human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G(919-1207) to support endothelial cell adhesion. In particular, endothelial cell adhesion to G(919-1207) is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016-1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins alphavbeta3 and beta1 and a combination of antibodies against alpha3 and alpha6 integrin subunits inhibit endothelial cell attachment to G(919-1207). Moreover, both alphavbeta3 and alpha3beta1 integrin bind with high affinity to G(919-1207). Together, our studies demonstrate that the G domain of laminin alpha4 chain is a specific, high affinity ligand for the alphavbeta3 and alpha3beta1 integrin heterodimers and that these integrins, together with alpha6beta1, function cooperatively to mediate endothelial cell-alpha4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the alphavbeta3 integrin in angiogenesis.     

Biodegradable dendritic positron-emitting nanoprobes for the noninvasive imaging of angiogenesis

A biodegradable positron-emitting dendritic nanoprobe targeted at α_vβ₃ integrin, a biological marker known to modulate angiogenesis, was developed for the noninvasive imaging of angiogenesis. The nanoprobe has a modular multivalent core-shell architecture consisting of a biodegradable heterobifunctional dendritic core chemoselectively functionalized with heterobifunctional polyethylene oxide (PEO) chains that form a protective shell, which imparts biological stealth and dictates the pharmacokinetics. Each of the 8 branches of the dendritic core was functionalized for labeling with radiohalogens. Placement of radioactive moieties at the core was designed to prevent in vivo dehalogenation, a potential problem for radiohalogens in imaging and therapy. Targeting peptides of cyclic arginine–glycine–aspartic acid (RGD) motifs were installed at the terminal ends of the PEO chains to enhance their accessibility to α_vβ₃ integrin receptors. This nanoscale design enabled a 50-fold enhancement of the binding affinity to α_vβ₃ integrin receptors with respect to the monovalent RGD peptide alone, from 10.40 nM to 0.18 nM IC₅₀. Cell-based assays of the ¹²⁵I-labeled dendritic nanoprobes using α_vβ₃-positive cells showed a 6-fold increase in α_vβ₃ receptor-mediated endocytosis of the targeted nanoprobe compared with the nontargeted nanoprobe, whereas α_vβ₃-negative cells showed no enhancement of cell uptake over time. In vivo biodistribution studies of ⁷⁶Br-labeled dendritic nanoprobes showed excellent bioavailability for the targeted and nontargeted nanoprobes. In vivo studies in a murine hindlimb ischemia model for angiogenesis revealed high specific accumulation of ⁷⁶Br-labeled dendritic nanoprobes targeted at α_vβ₃ integrins in angiogenic muscles, allowing highly selective imaging of this critically important process.     

Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of α4 integrin- and CXCR4-mediated engraftment in NOD/SCID β2-microglobulin-null mice

Background

Several studies indicate that ex vivo cytokine-supported expansion induces defective hematopoietic stem cell engraftment. We investigated the role of α4 integrin, α5 integrin and CXCR4 in engraftment of unmanipulated and cytokine-treated human cord blood CD34⁺ cells.

Design and Methods

Uncultured or expanded CD34⁺ cells were infused in NOD/SCID-β₂microglobulin-null mice. The function of α4, and α5 integrins and CXCR4 was assessed by incubating cells with specific neutralizing antibodies, prior to transplant. The activation state of α4 integrin was further tested by adhesion and migration assays.

Results

Neutralization of either α4 integrin or CXCR4 abolished engraftment of uncultured CD34⁺ cells at 6 week spost-transplant, while α5 integrin neutralization had no significant effect. However, after short-term ex vivo culture, blocking α4 integrin or CXCR4 did not affect repopulating activity whereas neutralization of α5 integrin inhibited engraftment. Using soluble vascular cell adhesion molecule-1 binding assays, we observed that α4 integrin affinity in fresh CD34⁺ cells was low and susceptible to stimulation while in cultured CD34⁺ cells, it was high and insensitive to further activation. In addition, stromal cell-derived factor-1 stimulated migration across vascular cell adhesion molecule-1 in fresh CD34⁺ cells but not in cultured CD34⁺ cells.

Conclusions

Our data show that ex vivo culture of hematopoietic progenitor cells is associated with downregulation of both α4 integrin- and CXCR4-mediated engraftment. Further investigations suggest that this is caused by supraphysiological increase of α4 integrin affinity, which impairs directional migration across vascular cell adhesion molecule-1 in response to stromal cell-derived factor-1. Such changes may underlie the engraftment defect of cytokine-stimulated CD34⁺ cells.