Tumor cell vascular mimicry: Novel targeting opportunity in melanoma.

In 1999, the American Journal of Pathology published an article, entitled "Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry" by Maniotis and colleagues, which ignited a spirited debate for several years and earned the journal's distinction of a "citation classic" (Maniotis et al., 1999). Tumor cell vasculogenic mimicry (VM), also known as vascular mimicry, describes the plasticity of aggressive cancer cells forming de novo vascular networks and is associated with the malignant phenotype and poor clinical outcome. The tumor cells capable of VM share the commonality of a stem cell-like, transendothelial phenotype, which may be induced by hypoxia. Since its introduction as a novel paradigm for melanoma tumor perfusion, many studies have contributed new findings illuminating the underlying molecular pathways supporting VM in a variety of tumors, including carcinomas, sarcomas, glioblastomas, astrocytomas, and melanomas. Of special significance is the lack of effectiveness of angiogenesis inhibitors on tumor cell VM, suggesting a selective resistance by this phenotype to conventional therapy. Facilitating the functional plasticity of tumor cell VM are key proteins associated with vascular, stem cell, extracellular matrix, and hypoxia-related signaling pathways--each deserving serious consideration as potential therapeutic targets and diagnostic indicators of the aggressive, metastatic phenotype. This review highlights seminal findings pertinent to VM, including the effects of a novel, small molecular compound, CVM-1118, currently under clinical development to target VM, and illuminates important molecular pathways involved in the suppression of this plastic, aggressive phenotype, using melanoma as a model.

Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α.

The activity of persistent Ca²âº sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca²âº entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca²âº sparklets by PKC-α and c-Src, both of which increase whole cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca²âº sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca²âº sparklets? With the use of total internal reflection fluorescence microscopy, Ca²âº sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Cav1.2c (the neuronal isoform of Cav1.2) constructs ± active or inactive PKC-α/c-Src. Cells expressing Cav1.2c exhibited both low-activity and persistent Ca²âº sparklets. Persistent Ca²âº sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Cav1.2c constructs mutated at one of two COOH-terminal residues (Y²¹²²F and Y²¹³9F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y²¹²²F but not Y²¹³9F Cav1.2c abrogated the potentiating effect of c-Src on Ca²âº sparklet activity. We could not detect a significant change in persistent Ca²âº sparklet activity or density in cells coexpressing Cav1.2c + PKC-α, regardless of whether WT or Y²¹²²F Cav1.2c was used, or after PP2 application, suggesting that PKC-α does not act upstream of c-Src to produce persistent Ca²âº sparklets. However, our results indicate that persistent Ca²âº sparklet activity is promoted by the action of c-Src on residue Y²¹²² of the Cav1.2c COOH terminus.

The roles of integrins in mediating the effects of mechanical force and growth factors on blood vessels in hypertension.

Hypertension is characterized by a sustained increase in vasoconstriction and attenuated vasodilation in the face of elevated mechanical stress in the blood vessel wall. To adapt to the increased stress, the vascular smooth muscle cell and its surrounding environment undergo structural and functional changes known as vascular remodeling. Multiple mechanisms underlie the remodeling process, including increased expression of humoral factors and their receptors as well as adhesion molecules and their receptors, all of which appear to collaborate and interact in the response to pressure elevation. In this review, we focus on the interactions between integrin signaling pathways and the activation of growth factor receptors in the response to the increased mechanical stress experienced by blood vessels in hypertension.

Spatial association of the Cav1.2 calcium channel with α5ß1-integrin.

Engagement of α(5)ß(1)-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (Ca(L)) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing Ca(L). Using coimmunoprecipitation strategies, we show that coassociation of Ca(L) with α(5)- or ß(1)-integrin in HEK293-T cells is specific and depends on cell adhesion to FN. In rat arteriolar smooth muscle, coassociations between Ca(L) and α(5)ß(1)-integrin and between Ca(L) and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of Ca(L) heterologously expressed in HEK293-T cells, we identified two regions of Ca(L) required for these interactions: 1) COOH-terminal residues Ser(1901) and Tyr(2122), known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively; and 2) two proline-rich domains (PRDs) near the middle of the COOH terminus. Immunofluorescence confocal imaging revealed a moderate degree of wild-type Ca(L) colocalization with ß(1)-integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, Ca(L) associates with α(5)ß(1)-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of Ca(L) at Y(2122) and/or S(1901) is required for association of Ca(L) with α(5)ß(1)-integrin; and 3) c-Src, via binding to PRDs that reside in the II-III linker region and/or the COOH terminus of Ca(L), mediates current potentiation following α(5)ß(1)-integrin engagement. These findings provide new evidence for how interactions between α(5)ß(1)-integrin and FN can modulate Ca(L) entry and consequently alter the physiological function of multiple types of excitable cells.

Coordinated regulation of vascular Ca2+ and K+ channels by integrin signaling.

A role for integrins in mechanotransduction has been suggested because these molecules form an important mechanical link between the extracellular matrix (ECM) and the cytoskeleton. An example of mechanotransduction in blood vessels is the myogenic response--the rapid and maintained constriction of arterioles in response to pressure elevation. L-type calcium channels and large-conductance, calcium-activated potassium (BK) channels are known to play important roles in the myogenic response and in the maintenance of myogenic (pressure-induced) vascular tone. Our recent studies on isolated, cannulated arterioles and freshly-dispersed arteriolar smooth muscle cells show that both L-type calcium channels (Ca(v)1.2) and BK channels are regulated by alpha5beta1 integrin activation. Alpha5beta1 integrin interacts with the ECM protein fibronectin, which is distributed in basement membrane and interstitial matrices surrounding smooth muscle cells within the arteriolar wall. Truncation and site-directed mutagenesis strategies reveal that regulation of Ca(v)1.2 by alpha5beta1 integrin requires phosphorylation of the channel alpha1C subunit at C-terminal residues Ser-1901 and Tyr-2122. Likewise, BK channel potentiation by alpha5beta1 integrin activation requires c-Src phosphorylation of the channel alpha-subunit at residue Tyr-766. Thus, both L-type calcium channels and BK channels can be regulated coordinately through integrin-linked phosphorylation cascades involving c-Src. We propose that these two channels are under constitutive control by alpha5beta1 integrin-fibronectin interactions in the vessel wall such that the balance of their activity determines myogenic tone and the vascular response to vessel wall injury/remodeling.

Modulation of alpha7-integrin-mediated adhesion and expression by platelet-derived growth factor in vascular smooth muscle cells.

We showed previously that the expression of alpha(7)-integrin in aortic vascular smooth muscle cells (VSMC) is enhanced in a rat model of atherosclerosis. In the present study, we investigated the effects of platelet-derived growth factor (PDGF) on alpha(7)-integrin expression and VSMC adhesion and migration. Expression of the alpha(7)-integrin gene was determined by real-time RT-PCR, whereas protein levels were determined by fluorescence-activated cell sorting analysis. PDGF increased alpha(7) cell surface protein expression (12 and 24 h: 3.3 +/- 0.8- and 3.6 +/- 0.4-fold, P < 0.05 vs. control) and mRNA levels (24 h: 3.1-fold, P < 0.05 vs. control) in a time-dependent manner. Actinomycin D and cycloheximide attenuated PDGF-induced increases in alpha(7)-integrin, indicating the involvement of de novo mRNA and protein synthesis. Treatment with the MAPK inhibitors PD-98059, SP-600125, and SB-203580 attenuated PDGF-induced increases in mRNA. In contrast, PD-98059 and SP-600125, but not SB-203580, attenuated PDGF-induced increases in cell surface protein levels. PDGF-treated VSMC adhered to laminin more efficiently (42 +/- 6% increase, P < 0.01), and this increase was partially inhibited by anti-alpha(7)-integrin function-blocking antibody. However, PDGF did not alter migration on laminin, and there was no effect of the anti-alpha(7)-integrin function-blocking antibody on basal or PDGF-stimulated migration. Immunofluorescence imaging revealed an increase in alpha(7)-integrin distribution along the stress fibers. Together, these observations indicate that PDGF enhances alpha(7)-integrin expression in VSMC and promotes alpha(7)-integrin-mediated adhesion to laminin.

Modulation of alpha4 integrin mRNA levels is coupled to deficits in vasomotor function in rat arterioles by allylamine.

Allylamine, a selective cardiovascular toxin that induces oxidative stress, is known to alter expression of extracellular matrix and cell adhesion proteins that are central to arterial remodeling. Our goals were to determine whether AAM treatment in rats modulates integrin/matrix-dependent arteriolar function, and to what extent integrin expression correlated to these alterations. Integrins are transmembrane proteins that facilitate mechanical and molecular signaling between the extracellular matrix and cytoskeleton, and so are suitable candidates for involvement in phenotypic and functional alterations of smooth muscle in response to oxidative stress. Arg-Gly-Asp (RGD) and Leu-Asp-Val (LDV), two integrin-binding motifs found in ECM proteins such as collagens and fibronectin, are known to interact with integrins alphavbeta3 and alpha4beta1, respectively. Previously, we found that RGD containing peptides induce vasodilation through alphavbeta3, while LDV containing peptides induce vasoconstriction through alpha4beta1 of normal rat cremasteric arterioles. In allylamine-treated rats (AAM), the vasomotor response to LDV, but not RGD, was attenuated in a dose-dependent manner. To determine whether changes in integrin subunit mRNA levels correlated with these functional changes, we performed reverse transcription and Real-time PCR for alpha4 and beta3 integrin subunits on RNA isolated from single, first-order cremasteric arterioles. AAM treatment caused a dose-dependent decrease in alpha4 mRNA expression, but not beta3 mRNA expression, suggesting that the changes in vasomotor activity to LDV peptides may be attributable in part to reduced alpha4 expression upon exposure to AAM. These data are supported by similar decreases in alpha4integrin cell surface protein expression in cultured vascular smooth muscle cells treated either in vivo and in vitro with AAM.

Modulation of cyclin dependent kinase inhibitor proteins and ERK1/2 activity in allylamine-injured vascular smooth muscle cells.

Chronic oxidative injury by allylamine (AAM) induces proliferative vascular smooth muscle cell (vSMC) phenotypes in the rat aorta similar to those seen in rodent and human atherosclerotic lesions. The proliferative advantage of AAM vSMC compared to control cells is maintained with serial passage of the cells and the advantage is nullified when AAM cells are seeded on a collagen substrate. In this study, we evaluate the potential role of cyclin dependent kinase inhibitors, p27 and p21, and mitogen activated protein (MAP) kinases, ERK1/2, in mediating the proliferative advantage of AAM stressed vSMC over control cells on plastic or collagen substrates. p27 levels in randomly cycling cells were comparable in both cell types irrespective of the substrate. In contrast, basal levels of p21 were 1.9 +/- 0.3 (P < 0.05)-fold higher in randomly cycling AAM cells seeded on plastic compared to controls, a difference that was lost on a collagen substrate. Following G0 synchronization, basal levels of both p27 and p21 were higher in AAM cells seeded on plastic compared to controls (1.7 +/- 0.2 and 2.0 +/- 0.3-fold, respectively, P < 0.05), but these differences were lost upon mitogenic stimulation. Pyrrolidine dithiocarbamate (PDTC) decreased p27 and p21 levels in cycling AAM cells relative to controls in a substrate-dependent manner. AAM cells seeded on plastic exhibited enhanced ERK1/2 activation upon mitogenic stimulation; seeding on collagen nullified this advantage. The duration of ERK1/2 activation was prolonged in AAM cells independently of the seeding substrate. We conclude that substrate-dependent acquisition of proliferative phenotypes following repeated cycles of AAM injury correlates with modulation of the cyclin dependent kinase inhibitors, p27 and p21.

Regulation of alpha7-integrin expression in vascular smooth muscle by injury-induced atherosclerosis.

Injury of vascular smooth muscle cells (VSMCs) by allylamine (AAM) leads to phenotypic changes associated with atherogenic progression including increased proliferation, migration, and alterations in cell adhesion. In the present study, the relationship between AAM-induced vascular injury and expression of the alpha(7)-integrin subunit was investigated. The alpha(7)-mRNA and protein expression were examined using real-time RT-PCR, fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and immunoblotting. In cultured VSMCs from aortas of AAM-treated rats (70 mg/kg for 20 days), alpha(7)-mRNA levels were increased more than twofold compared with control cells. No change was seen in beta(1)-integrin expression. FACS analysis revealed increased cell surface expression of alpha(7)-protein (25 +/- 9%; *P < 0.05). AAM treatment of naive VSMCs enhanced alpha(7)-mRNA expression (2.4 +/- 0.7-fold, mean +/- SE; *P < 0.05). The increased alpha(7)-mRNA expression was attenuated by the amine oxidase inhibitor semicarbazide and the antioxidant pyrrolidine dithiocarbamate, which confirms a role for oxidative stress in modulating alpha(7)-expression. In vivo alpha(7)-mRNA and protein expression were enhanced in the aortas of AAM-treated rats. In addition, increased alpha(7)-integrin expression facilitated AAM VSMC adhesion to laminin more efficiently compared with control (51 +/- 2%; *P < 0.05). Chemical injury induced by AAM significantly enhances alpha(7)-integrin expression in VSMCs. These findings implicate for the first time the expression of alpha(7)-integrin during the response of VSMCs to vascular injury.

Human serum albumin and its structural variants mediate cholesterol efflux from cultured endothelial cells.

In the present study, we used the human EA.hy926 endothelial cell line as the model system to investigate the effect of human serum albumin (HSA) and its structural variants on cholesterol efflux. Initial studies showed that HSA promoted cholesterol efflux in a dose- and time-dependent manner, reaching a plateau at 10 mg/ml at 90 min. As a control, gelatin displayed no significant effect on efflux, while HSA was significantly more efficient than ovalbumin and bovine serum albumin (BSA) in promoting cholesterol efflux. Equal molar concentrations of HSA and apolipoprotein A-I (apoA-I) showed that apoA-I had considerably higher efficiency in efflux. However, the prevailing high plasma concentrations of HSA may compensate for its lower efflux rate compared to apoA-I. To characterize the mechanism of HSA-mediated cholesterol efflux, we studied the effects of cAMP and temperature on efflux using both EA.hy926 endothelial cells and murine RAW 264.7 macrophages. We found that HSA-mediated efflux occurred via a cAMP-independent and relatively temperature-insensitive pathway. We next examined the nature of HSA-cholesterol interaction by comparing the effects of various HSA mutants to wild-type HSA on cholesterol efflux. We found specific interactions between subdomains 2A and 3A and cholesterol, as indicated by the changes in the efflux rate of various HSA mutants. In conclusion, our study provides evidence for the role of HSA in cholesterol efflux, and shows that the substitution of specific amino acid residues in subdomains of 2A and 3A may be important structural determinants in its ability to bind to cholesterol and participate in cholesterol efflux.

Inhibitory effect of delta-tocotrienol, a HMG CoA reductase inhibitor, on monocyte-endothelial cell adhesion.

We have previously shown that alpha-tocotrienol (alpha-T3), a vitamin E analogue and HMG CoA reductase (HMGR) inhibitor, markedly inhibited monocyte-endothelial cell adhesion, a process that was reversed with the addition of mevalonate intermediates involved in protein prenylation. Since delta-T3 and gamma-T3 possess greater HMGR inhibition than alpha-T3, we postulated that these analogues might have a greater effect on protein prenylation, and thus on monocyte adhesion and endothelial adhesion molecule expression in comparison to alpha-T3. Hence, we pursued to investigate the effect of various analogues of tocotrienol (alpha, gamma, delta) on monocytic cell adhesion and expression of adhesion molecules using a human umbilical vein endothelial cell-line, EA.hy926, as the model system. Relative to alpha-T3, delta-T3 displayed a more profound inhibitory effect on monocytic cell adherence using a 15 micromol/L concentration within 24 h (delta: 42 +/- 5%; alpha: 26 +/- 8% vs. control). This inhibitory action was reversed by co-incubation with farnesol and geranylgeraniol, suggesting a role for prenylated proteins in the regulation of monocyte adhesion. To further evaluate the effect of tocotrienols on the vascular endothelium, we measured the surface expression of adhesion molecules. Compared to alpha-T3, delta-T3 markedly inhibited the expression of VCAM-1 (delta: 57 +/- 6%; alpha: 37 +/- 10% vs. control) and E-selection (delta: 36 +/- 3%; alpha: 18 +/- 6% vs. control) in TNF-alpha activated endothelial cells. The above result suggests that delta-T3 is a potent and effective agent for the reduction of cellular adhesion molecule expression and monocytic cell adherence.