Laura di Blasio

Identification of CD36 molecular features required for its in vitro angiostatic activity.

Thrombospondin‐1 (TSP‐1), a natural inhibitor of angiogenesis, acts directly on endothelial cells (EC) via CD36 to inhibit their migration and morphogenesis induced by basic fibroblast growth factor. Here we show that CD36 triggered by TSP‐1 inhibits in vitro angiogenesis stimulated by vascular endothelial growth factor‐A (VEGF‐A). To demonstrate that the TSP‐1 inhibitory signal was mediated by CD36, we transduced CD36 in CD36‐deficient endothelial cells. Both TSP‐1 and the agonist anti‐CD36 mAb SMO, which mimics TSP‐1 activity, reduced the VEGF‐A165‐induced migration and sprouting of CD36‐ECs. To address the mechanisms by which CD36 may exert its angiostatic function, we investigated the functional components of the C‐terminal cytoplasmic tail by site‐directed mutagenesis. Our results indicate that C464, R467, and K469 of CD36 are required for the inhibitory activity of TSP‐1. In contrast, point mutation of C466 did not alter TSP‐1 ability to inhibit EC migration and sprouting. Moreover, we show that activation of CD36 by TSP‐1 down‐modulates the VEGF receptor‐2 (VEGFR‐2) and p38 mitogen‐associated protein kinase phosphorylation induced by VEGF‐A165, and this effect was specifically abolished by point mutation at C464. These results identify specific amino acids of the C‐terminal cytoplasmic tail of CD36 crucial for the in vitro angiostatic activity of TSP‐1 and extend our knowledge of regulation of VEGFR‐2‐mediated biological activities on ECs.

Essential role of PDK1 in regulating endothelial cell migration

The serine/threonine protein kinase phosphoinositide-dependent kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases, including PKB/Akt. We now present evidence showing that PDK1 is essential for the motility of vascular endothelial cells (ECs) and that it is involved in the regulation of their chemotaxis. ECs differentiated from mouse embryonic stem cells lacking PDK1 completely lost their ability to migrate in vitro in response to vascular endothelial growth factor-A (VEGF-A). In addition, PDK1−/− embryoid bodies exhibit evident developmental and vascular defects that can be attributed to a reduced cell migration. Moreover, the overexpression of PDK1 increased the EC migration induced by VEGF-A. We propose a model of spatial distribution of PDK1 and Akt in which the synthesis of phosphatidylinositol 3,4,5 triphosphate at plasma membrane by activation of phosphoinositide 3-kinase recruits both proteins at the leading edge of the polarized ECs and promotes cell chemotaxis. These findings establish a mechanism for the spatial localization of PDK1 and its substrate Akt to regulate directional migration.

Modeling human tumor angiogenesis in a three-dimensional culture system

The intrinsic complexity of the process of vessel formation limits the efficacy of cellular assays for elucidation of its molecular and pharmacologic mechanisms. We developed an ex vivo three-dimensional (3D) assay of sprouting angiogenesis with arterial explants from human umbilical cords. In this assay, human arterial rings were embedded in basement membrane extract gel, leading to a network of capillarylike structures upon vascular endothelial growth factor (VEGF) A stimulation. The angiogenic outgrowth consisted of endothelial cells, which actively internalized acetylated-low-density lipoprotein, surrounded by pericytes. Computer-assisted quantification of this vascular network demonstrated considerable sensitivity of this assay to several angiogenic inhibitors, including kinase inhibitors and monoclonal antibodies. We also performed targeted gene knockdown on this model by directly infecting explanted umbilical arteries with lentiviruses carrying short-hairpin RNA. Downregulation of VEGFR2 resulted in a significant reduction of the sprouting capability, demonstrating the relevance of human vascular explants for functional genomics studies. Furthermore, a modification of this assay led to development of a 3D model of tumor-driven angiogenesis, in which angiogenic outgrowth was sustained by spheroids of prostate cancer cells in absence of exogenous growth factors. The human arterial ring assay bridges the gap between in vitro endothelial cell and animal model, and is a powerful system for identification of genes and drugs that regulate human angiogenesis.

The miR-126 regulates angiopoietin-1 signaling and vessel maturation by targeting p85β.

Blood vessel formation depends on the highly coordinated actions of a variety of angiogenic regulators. Vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1) are both potent and essential proangiogenic factors with complementary roles in vascular development and function. Whereas VEGF is required for the formation of the initial vascular plexus, Ang-1 contributes to the stabilization and maturation of growing blood vessels. Here, we provide evidence of a novel microRNA (miRNA)-dependent molecular mechanism of Ang-1 signalling modulation aimed at stabilizing adult vasculature. MiRNAs are short non-coding RNA molecules that post-trascriptionally regulate gene expression by translational suppression or in some instances by cleavage of the respective mRNA target. Our data indicate that endothelial cells of mature vessels express high levels of miR-126, which primarily targets phosphoinositide-3-kinase regulatory subunit 2 (p85β). Down-regulation of miR-126 and over-expression of p85β in endothelial cells inhibit the biological functions of Ang-1. Additionally, knockdown of miR-126 in zebrafish resulted in vascular remodelling and maturation defects, reminiscent of the Ang-1 loss-of-function phenotype. Our findings suggest that miR-126-mediated phosphoinositide-3-kinase regulation, not only fine-tunes VEGF-signaling, but it strongly enhances the activities of Ang-1 on vessel stabilization and maturation.

Protein Kinase D1 Regulates VEGF-A-Induced αvβ3 Integrin Trafficking and Endothelial Cell Migration

The bidirectional communication between integrin αvβ3 and vascular endothelial growth factor (VEGF) receptors acts to integrate and coordinate endothelial cell (EC) activity during angiogenesis. However, the molecular mechanisms involved in this signaling crosstalk are only partially revealed. We have found that protein kinase D1 (PKD1) was activated by VEGF‐A, but not by other angiogenic factors, and associated with αvβ3 integrin. Moreover, knockdown of PKD1 increased endocytosis of αvβ3 and reduced its return from endosomes to the plasma membrane leading to accumulation of the integrin in Rab5‐ and Rab4‐positive endosomes. Consistent with this, PKD1 knockdown caused defects in focal complex formation and reduced EC migration in response to VEGF‐A. Moreover, knockdown of PKD1 reduced EC motility on vitronectin, whereas migration on collagen I was not PKD1 dependent. These results suggest that PKD1‐regulated αvβ3 trafficking contributes to the angiogenesis process by integrating VEGF‐A signaling with extracellular matrix interactions.

Increased Expression of α6 Integrin in Endothelial Cells Unveils a Proangiogenic Role for Basement Membrane

The integrin alpha6 subunit is part of the alpha6beta1 and alpha6beta4 integrin complexes, which are known to be receptors for laminins and to mediate several biological activities such as embryogenesis, organogenesis, and invasion of carcinoma cells. However, the precise role of alpha6 integrin in angiogenesis has not yet been addressed. We observed that both vascular endothelial growth factor-A and fibroblast growth factor-2 strongly upregulate alpha6 integrin in human endothelial cells. Moreover, alpha6 integrin was positively modulated in angiogenic vessels in pancreatic neuroendocrine carcinoma. In this transgenic mouse model of spontaneous tumorigenesis, alpha6 integrin expression increased in the angiogenic stage, while being expressed at low levels in normal and hyperplastic tissue. We studied the functional role of alpha6 integrin during angiogenesis by lentivirus-mediated gene silencing and blocking antibody. Cell migration and morphogenesis on basement membrane extracts, a laminin-rich matrix, was reduced in endothelial cells expressing low levels of alpha6 integrin. However, we did not observe any differences in collagen matrices. Similar results were obtained in the aortic ring angiogenesis assay. alpha6 integrin was required for vessel sprouting on basement membrane gels but not on collagen gels, as shown by stably silencing this integrin in the murine aorta. Finally, a neutralizing anti-alpha6 integrin antibody inhibited in vivo angiogenesis in chicken chorioallantoic membrane and transgenic tumor mouse model. In summary, we showed that the alpha6 integrin participated in vascular endothelial growth factor-A and fibroblast growth factor-2-driven angiogenesis in vitro and in vivo, suggesting that it might be an attractive target for therapeutic approaches in angiogenesis-dependent diseases such as tumor growth.

PDK1: A signaling hub for cell migration and tumor invasion

The ability of cells to migrate is essential for different physiological processes including embryonic development, angiogenesis, tissue repair and immune response. In the context of cancer such abilities acquire dramatic implications, as they are exploited by tumor cells to invade neighboring or distant healthy tissues. 3-Phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1) is an ancient serine-threonine kinase belonging to AGC kinase family. An increasing amount of data points at a pivotal role for PDK1 in the regulation of cell migration. PDK1 is a transducer of PI3K signaling and activates multiple downstream effectors, thereby representing an essential hub coordinating signals coming from extracellular cues to the cytoskeletal machinery, the final executor of cell movement. Akt, PAK1, β3 integrin, ROCK1, MRCKα and PLCγ1 are, according to the literature, the signaling transducers through which PDK1 regulates cell migration. In addition, PDK1 contributes to tumor cell invasion by regulating invadopodia formation and both amoeboid and collective cancer cell invasion. This and other pieces of evidence, such as its reported overexpression across several tumor types, corroborate a PDK1 role tumor aggressiveness. Altogether, these findings indicate the possibility to rationally target PDK1 in human tumors in order to counteract cancer cell dissemination in the organism.

PDK1-mediated activation of MRCKα regulates directional cell migration and lamellipodia retraction

MRCKα is activated by PDK1 through a PIP3-dependent, kinase-independent mechanism that drives the relocation of both proteins to lamellipodia and regulates lamellipodial retraction and directional migration.

Serine/Threonine Kinase 3-Phosphoinositide-Dependent Protein Kinase-1 (PDK1) as a Key Regulator of Cell Migration and Cancer Dissemination

Dissecting the cellular signaling that governs the motility of eukaryotic cells is one of the fundamental tasks of modern cell biology, not only because of the large number of physiological processes in which cell migration is crucial, but even more so because of the pathological ones, in particular tumor invasion and metastasis. Cell migration requires the coordination of at least four major processes: polarization of intracellular signaling, regulation of the actin cytoskeleton and membrane extension, focal adhesion and integrin signaling and contractile forces generation and rear retraction. Among the molecular components involved in the regulation of locomotion, the phosphatidylinositol-3-kinase (PI3K) pathway has been shown to exert fundamental role. A pivotal node of such pathway is represented by the serine/threonine kinase 3-phosphoinositide-dependent protein kinase-1 (PDPK1 or PDK1). PDK1, and the majority of its substrates, belong to the AGC family of kinases (related to cAMP-dependent protein kinase 1, cyclic Guanosine monophosphate-dependent protein kinase and protein kinase C), and control a plethora of cellular processes, downstream either to PI3K or to other pathways, such as RAS GTPase-MAPK (mitogen-activated protein kinase). Interestingly, PDK1 has been demonstrated to be crucial for the regulation of each step of cell migration, by activating several proteins such as protein kinase B/Akt (PKB/Akt), myotonic dystrophy-related CDC42-binding kinases alpha (MRCKα), Rho associated coiled-coil containing protein kinase 1 (ROCK1), phospholipase C gamma 1 (PLCγ1) and β3 integrin. Moreover, PDK1 regulates cancer cell invasion as well, thus representing a possible target to prevent cancer metastasis in human patients. The aim of this review is to summarize the various mechanisms by which PDK1 controls the cell migration process, from cell polarization to actin cytoskeleton and focal adhesion regulation, and finally, to discuss the evidence supporting a role for PDK1 in cancer cell invasion and dissemination.

Real-time monitoring of cell protrusion dynamics by impedance responses

Cellular protrusions are highly dynamic structures involved in fundamental processes, including cell migration and invasion. For a cell to migrate, its leading edge must form protrusions, and then adhere or retract. The spatial and temporal coordination of protrusions and retraction is yet to be fully understood. The study of protrusion dynamics mainly relies on live-microscopy often coupled to fluorescent labeling. Here we report the use of an alternative, label-free, quantitative and rapid assay to analyze protrusion dynamics in a cell population based on the real-time recording of cell activity by means of electronic sensors. Cells are seeded on a plate covered with electrodes and their shape changes map into measured impedance variations. Upon growth factor stimulation the impedance increases due to protrusive activity and decreases following retraction. Compared to microscopy-based methods, impedance measurements are suitable to high-throughput studies on different cell lines, growth factors and chemical compounds. We present data indicating that this assay lends itself to dissect the biochemical signaling pathways controlling adhesive protrusions. Indeed, we show that the protrusion phase is sustained by actin polymerization, directly driven by growth factor stimulation. Contraction instead mainly relies on myosin action, pointing at a pivotal role of myosin in lamellipodia retraction.