Vicrostatin – An Anti-Invasive Multi-Integrin Targeting Chimeric Disintegrin with Tumor Anti-Angiogenic and Pro-Apoptotic Activities

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., αvβ3, αvβ5, and α5β1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN

Successful Inhibition of Tumor Development by Specific Class-3 Semaphorins Is Associated with Expression of Appropriate Semaphorin Receptors by Tumor Cells

The class-3 semaphorins (sema3s) include seven family members. Six of them bind to neuropilin-1 (np1) or neuropilin-2 (np2) receptors or to both, while the seventh, sema3E, binds to the plexin-D1 receptor. Sema3B and sema3F were previously characterized as tumor suppressors and as inhibitors of tumor angiogenesis. To determine if additional class-3 semaphorins such as sema3A, sema3D, sema3E and sema3G possess anti-angiogenic and anti-tumorigenic properties, we expressed the recombinant full length semaphorins in four different tumorigenic cell lines expressing different combinations of class-3 semaphorin receptors. We show for the first time that sema3A, sema3D, sema3E and sema3G can function as potent anti-tumorigenic agents. All the semaphorins we examined were also able to reduce the concentration of tumor associated blood vessels although the potencies of the anti-angiogenic effects varied depending on the tumor cell type. Surprisingly, there was little correlation between the ability to inhibit tumor angiogenesis and their anti-tumorigenic activity. None of the semaphorins inhibited the adhesion of the tumor cells to plastic or fibronectin nor did they modulate the proliferation of tumor cells cultured in cell culture dishes. However, various semaphorins were able to inhibit the formation of soft agar colonies from tumor cells expressing appropriate semaphorin receptors, although in this case too the inhibitory effect was not always correlated with the anti-tumorigenic effect. In contrast, the anti-tumorigenic effect of each of the semaphorins correlated very well with tumor cell expression of specific signal transducing receptors for particular semaphorins. This correlation was not broken even in cases in which the tumor cells expressed significant concentrations of endogenous semaphorins. Our results suggest that combinations of different class-3 semaphorins may be more effective than single semaphorins in cases in which tumor cells express more than one type of semaphorin receptors

Reversal of TGF-β1 stimulation of α-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's - derived fibroblasts

<p>Abstract</p> <p>Background</p> <p>Myofibroblasts, a derived subset of fibroblasts especially important in scar formation and wound contraction, have been found at elevated levels in affected Dupuytren's tissues. Transformation of fibroblasts to myofibroblasts is characterized by expression of alpha- smooth muscle actin (α-SMA) and increased production of extracellular matrix (ECM) components, both events of relevance to connective tissue remodeling. We propose that increasing the activation of the cyclic AMP (cAMP)/protein kinase A signaling pathway will inhibit transforming growth factor-beta1 (TGF-β₁)-induced ECM synthesis and myofibroblast formation and may provide a means to blunt fibrosis.</p> <p>Methods</p> <p>Fibroblasts derived from areas of Dupuytren's contracture cord (DC), from adjacent and phenotypically normal palmar fascia (PF), and from palmar fascia from patients undergoing carpal tunnel release (CTR; CT) were treated with TGF-β₁(2 ng/ml) and/or forskolin (10 μM) (a known stimulator of cAMP). Total RNA and protein extracted was subjected to real time RT-PCR and Western blot analysis.</p> <p>Results</p> <p>The basal mRNA expression levels of fibronectin- extra domain A (FN1-EDA), type I (COL1A2) and type III collagen (COL3A1), and connective tissue growth factor (CTGF) were all significantly increased in DC- and in PF-derived cells compared to CT-derived fibroblasts. The TGF-β₁stimulation of α-SMA, CTGF, COL1A2 and COL3A1 was greatly inhibited by concomitant treatment with forskolin, especially in DC-derived cells. In contrast, TGF-β₁stimulation of FN1-EDA showed similar levels of reduction with the addition of forskolin in all three cell types.</p> <p>Conclusion</p> <p>In sum, increasing cAMP levels show potential to inhibit the formation of myofibroblasts and accumulation of ECM components. Molecular agents that increase cAMP may therefore prove useful in mitigating DC progression or recurrence.</p

Integrin β3 Crosstalk with VEGFR Accommodating Tyrosine Phosphorylation as a Regulatory Switch

Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between β3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of β3 and VEGFR2. Specifically, the membrane-proximal motif around 801YLSI in VEGFR2 mediates its binding to non-phosphorylated β3CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y747 phosphorylation of β3 enhances the above interaction. To demonstrate the importance of β3 phosphorylation in endothelial cell functions, we synthesized β3CT-mimicking Y747 phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y747 but not F747 significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y747 peptide exhibits inhibitory effect only in WT but not in β3 integrin knock-out or β3 integrin knock-in cells expressing β3 with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2

PDGF-Rα gene expression predicts proliferation, but PDGF-A suppresses transdifferentiation of neonatal mouse lung myofibroblasts

<p>Abstract</p> <p>Background</p> <p>Platelet-derived growth factor A (PDGF-A) signals solely through PDGF-Rα, and is required for fibroblast proliferation and transdifferentiation (fibroblast to myofibroblast conversion) during alveolar development, because <it>pdgfa</it>-null mice lack both myofibroblasts and alveoli. However, these PDGF-A-mediated mechanisms remain incompletely defined. At postnatal days 4 and 12 (P4 and P12), using mouse lung fibroblasts, we examined (a) how PDGF-Rα correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (αSMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects αSMA or modifies stimulation by transforming growth factor beta (TGFβ).</p> <p>Methods</p> <p>Using flow cytometry we examined PDGF-Rα, αSMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-Rα expression. Using real-time RT-PCR we quantified αSMA mRNA in cultured Mlg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGFβ.</p> <p>Results</p> <p>The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-Rα. At P4, more of the higher than lower PDGF-Rα + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the αSMA + but not the αSMA- population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-Rα + and αSMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-Rα + and αSMA- whereas at P12, most Ki67+ fibroblasts were PDGF-Rα- and αSMA-. More of the PDGF-Rα + than - fibroblasts contained αSMA at both P4 and P12. In the lung, proximate αSMA was more abundant around nuclei in cells expressing high than low levels of PDGF-Rα at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-Rα-, but not in PDGF-Rα + cells. In Mlg fibroblasts, αSMA mRNA increased after exposure to TGFβ, but declined after treatment with PDGF-A.</p> <p>Conclusion</p> <p>During both septal eruption (P4) and elongation (P12), alveolar PDGF-Rα may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate αSMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-Rα more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGFβ may overshadow the antagonistic effects of PDGF-A/PDGF-Rα signaling, enhancing αSMA-abundance in PDGF-Rα-expressing fibroblasts.</p

Docosahexaenoic Acid Inhibits UVB-Induced Activation of NF-κB and Expression of COX-2 and NOX-4 in HR-1 Hairless Mouse Skin by Blocking MSK1 Signaling

Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Docosahexaenoic acid (DHA), a representative ω-3 polyunsaturated fatty acid, has been reported to possess anti-inflammatory and chemopreventive properties. In the present study, we investigated the molecular mechanisms underlying the inhibitory effects of DHA on UVB-induced inflammation in mouse skin. Our study revealed that topical application of DHA prior to UVB irradiation attenuated the expression of cyclooxygenase-2 (COX-2) and NAD(P)H:oxidase-4 (NOX-4) in hairless mouse skin. DHA pretreatment also attenuated UVB-induced DNA binding of nuclear factor-kappaB (NF-κB) through the inhibition of phosphorylation of IκB kinase-α/β, phosphorylation and degradation of IκBα and nuclear translocation of p50 and p65. In addition, UVB-induced phosphorylation of p65 at the serine 276 residue was significantly inhibited by topical application of DHA. Irradiation with UVB induced phosphorylation of mitogen and stress-activated kinase-1 (MSK1), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, and all these events were attenuated by pretreatment with DHA. Blocking ERK and p38 MAP kinase signaling by U0126 and SB203580, respectively, diminished MSK1 phosphorylation in UVB-irradiated mouse skin. Pretreatment with H-89, a pharmacological inhibitor of MSK1, abrogated UVB-induced activation of NF-κB and the expression of COX-2 and NOX-4 in mouse skin. In conclusion, topically applied DHA inhibits the UVB-induced activation of NF-κB and the expression of COX-2 and NOX-4 by blocking the phosphorylation of MSK1, a kinase downstream of ERK and p38 MAP kinase, in hairless mouse skin

Topography of Extracellular Matrix Mediates Vascular Morphogenesis and Migration Speeds in Angiogenesis

The extracellular matrix plays a critical role in orchestrating the events necessary for wound healing, muscle repair, morphogenesis, new blood vessel growth, and cancer invasion. In this study, we investigate the influence of extracellular matrix topography on the coordination of multi-cellular interactions in the context of angiogenesis. To do this, we validate our spatio-temporal mathematical model of angiogenesis against empirical data, and within this framework, we vary the density of the matrix fibers to simulate different tissue environments and to explore the possibility of manipulating the extracellular matrix to achieve pro- and anti-angiogenic effects. The model predicts specific ranges of matrix fiber densities that maximize sprout extension speed, induce branching, or interrupt normal angiogenesis, which are independently confirmed by experiment. We then explore matrix fiber alignment as a key factor contributing to peak sprout velocities and in mediating cell shape and orientation. We also quantify the effects of proteolytic matrix degradation by the tip cell on sprout velocity and demonstrate that degradation promotes sprout growth at high matrix densities, but has an inhibitory effect at lower densities. Our results are discussed in the context of ECM targeted pro- and anti-angiogenic therapies that can be tested empirically

Early Embryonic Vascular Patterning by Matrix-Mediated Paracrine Signalling: A Mathematical Model Study

During embryonic vasculogenesis, endothelial precursor cells of mesodermal origin known as angioblasts assemble into a characteristic network pattern. Although a considerable amount of markers and signals involved in this process have been identified, the mechanisms underlying the coalescence of angioblasts into this reticular pattern remain unclear. Various recent studies hypothesize that autocrine regulation of the chemoattractant vascular endothelial growth factor (VEGF) is responsible for the formation of vascular networks in vitro. However, the autocrine regulation hypothesis does not fit well with reported data on in vivo early vascular development. In this study, we propose a mathematical model based on the alternative assumption that endodermal VEGF signalling activity, having a paracrine effect on adjacent angioblasts, is mediated by its binding to the extracellular matrix (ECM). Detailed morphometric analysis of simulated networks and images obtained from in vivo quail embryos reveals the model mimics the vascular patterns with high accuracy. These results show that paracrine signalling can result in the formation of fine-grained cellular networks when mediated by angioblast-produced ECM. This lends additional support to the theory that patterning during early vascular development in the vertebrate embryo is regulated by paracrine signalling

3D Multi-Cell Simulation of Tumor Growth and Angiogenesis

We present a 3D multi-cell simulation of a generic simplification of vascular tumor growth which can be easily extended and adapted to describe more specific vascular tumor types and host tissues. Initially, tumor cells proliferate as they take up the oxygen which the pre-existing vasculature supplies. The tumor grows exponentially. When the oxygen level drops below a threshold, the tumor cells become hypoxic and start secreting pro-angiogenic factors. At this stage, the tumor reaches a maximum diameter characteristic of an avascular tumor spheroid. The endothelial cells in the pre-existing vasculature respond to the pro-angiogenic factors both by chemotaxing towards higher concentrations of pro-angiogenic factors and by forming new blood vessels via angiogenesis. The tumor-induced vasculature increases the growth rate of the resulting vascularized solid tumor compared to an avascular tumor, allowing the tumor to grow beyond the spheroid in these linear-growth phases. First, in the linear-spherical phase of growth, the tumor remains spherical while its volume increases. Second, in the linear-cylindrical phase of growth the tumor elongates into a cylinder. Finally, in the linear-sheet phase of growth, tumor growth accelerates as the tumor changes from cylindrical to paddle-shaped. Substantial periods during which the tumor grows slowly or not at all separate the exponential from the linear-spherical and the linear-spherical from the linear-cylindrical growth phases. In contrast to other simulations in which avascular tumors remain spherical, our simulated avascular tumors form cylinders following the blood vessels, leading to a different distribution of hypoxic cells within the tumor. Our simulations cover time periods which are long enough to produce a range of biologically reasonable complex morphologies, allowing us to study how tumor-induced angiogenesis affects the growth rate, size and morphology of simulated tumors