Guido Serini

Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function

The motility and morphogenesis of endothelial cells is controlled by spatio-temporally regulated activation of integrin adhesion receptors, and integrin activation is stimulated by major determinants of vascular remodelling. In order for endothelial cells to be responsive to changes in activator gradients, the adhesiveness of these cells to the extracellular matrix must be dynamic, and negative regulators of integrins could be required. Here we show that during vascular development and experimental angiogenesis, endothelial cells generate autocrine chemorepulsive signals of class 3 semaphorins (SEMA3 proteins) that localize at nascent adhesive sites in spreading endothelial cells. Disrupting endogenous SEMA3 function in endothelial cells stimulates integrin-mediated adhesion and migration to extracellular matrices, whereas exogenous SEMA3 proteins antagonize integrin activation. Misexpression of dominant negative SEMA3 receptors in chick embryo endothelial cells locks integrins in an active conformation, and severely impairs vascular remodelling. Sema3a null mice show vascular defects as well. Thus during angiogenesis endothelial SEMA3 proteins endow the vascular system with the plasticity required for its reshaping by controlling integrin function.

Modeling the early stages of vascular network assembly

In vertebrates, networks of capillary vessels supply tissues with nutrients. Capillary patterns are closely mimicked by endothelial cells cultured on basement membrane proteins that allow single randomly dispersed cells to self‐organize into vascular networks. Here we provide a model including chemoattraction as the fundamental mechanism for cell‐to‐cell communication in order to identify key parameters in the complexity of the formation of vascular patterns. By flanking biological experiments, theoretical insights and numerical simulations, we provide strong evidence that endothelial cell number and the range of activity of a chemoattractant factor regulate vascular network formation and size. We propose a mechanism linking the scale of formed endothelial structures to the range of cell‐to‐cell interaction mediated by the release of chemoattractants.

Neuropilin-1/GIPC1 signaling regulates α5β1 integrin traffic and function in endothelial cells

Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes α5β1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with α5β1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active α5β1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with α5β1, and the associated motor myosin VI (Myo6) support active α5β1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active α5β1. Nrp1 modulation of α5β1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.

Percolation, Morphogenesis, and Burgers Dynamics in Blood Vessels Formation

Experiments of in vitro formation of blood vessels show that cells randomly spread on a gel matrix autonomously organize to form a connected vascular network. We propose a simple model which reproduces many features of the biological system. We show that both the model and the real system exhibit a fractal behavior at small scales, due to the process of migration and dynamical aggregation, followed at large scale by a random percolation behavior due to the coalescence of aggregates. The results are in good agreement with the analysis performed on the experimental data.

Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models

Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.

In vivo activation of JAK2/STAT-3 pathway during angiogenesis induced by GM-CSF

Besides the regulation of hematopoiesis, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) induces the expression of a functional program in cultured endothelial cells (ECs) related to angiogenesis and to the their survival in bone marrow microenvironment. ECs express the specific GM‐CSF receptor that signals through the recruitment and the activation of Janus kinase (JAK)2 (Soldi et al., Blood 89, 863–872, 1987). We now report that GM‐CSF in vivo induces angiogenesis and activates JAK‐2 and signal transducers and activators of transcription (STAT)‐3. This cytokine has an angiogenetic activity in chick chorioallantoic membrane (CAM) without recruitment of inflammatory cells and induces vessel sprouting from chicken aorta rings. When added to CAM, subnanomolar concentrations of GM‐CSF cause a rapid phosphorylation in tyrosine residues of JAK‐2 persisting at least for 10 min. Furthermore, we show that signal transducers and activators of transcription (STAT)‐3, but not STAT‐5, also are phosphorylated for 30 min after GM‐CSF stimulation. AG‐490, a JAK‐2 inhibitor, reduced in a dose‐dependent manner the angiogenic effect of GM‐CSF in CAM. These findings provide the first evidence that the JAK‐2/STAT‐3 pathway is activated in vivo and participates in vessel formation triggered by GM‐CSF.

Semaphorin 3A overcomes cancer hypoxia and metastatic dissemination induced by antiangiogenic treatment in mice

Cancer development, progression, and metastasis are highly dependent on angiogenesis. The use of antiangiogenic drugs has been proposed as a novel strategy to interfere with tumor growth, but cancer cells respond by developing strategies to escape these treatments. In particular, animal models show that antiangiogenic drugs currently used in clinical settings reduce tumor tissue oxygenation and trigger molecular events that foster cancer resistance to therapy. Here, we show that semaphorin 3A (Sema3A) expression overcomes the proinvasive and prometastatic resistance observed upon angiogenesis reduction by the small-molecule tyrosine inhibitor sunitinib in both pancreatic neuroendocrine tumors (PNETs) in RIP-Tag2 mice and cervical carcinomas in HPV16/E2 mice. By improving cancer tissue oxygenation and extending the normalization window, Sema3A counteracted sunitinib-induced activation of HIF-1α, Met tyrosine kinase receptor, epithelial-mesenchymal transition (EMT), and other hypoxia-dependent signaling pathways. Sema3A also reduced tumor hypoxia and halted cancer dissemination induced by DC101, a specific inhibitor of the VEGF pathway. As a result, reexpressing Sema3A in cancer cells converts metastatic PNETs and cervical carcinomas into benign lesions. We therefore suggest that this strategy could be developed to safely harnesses the therapeutic potential of the antiangiogenic treatment.

AP-2α and AP-2γ regulate tumor progression via specific genetic programs

The events occurring during tumor formation and progression display similarities to some of the steps in embryonic morphogenesis. The family of AP‐2 proteins consists of five different transcription factors (α, β, γ, δ, and ε) that play relevant roles in embryonic development, as demonstrated by the phenotypes of the corresponding knockout mice. Here, we show that AP‐2α and AP‐2γ proteins play an essential role in tumorigenesis. Down‐modulation of AP‐2 expression in tumor cells by RNA interference (RNAi) led to enhanced tumor growth and reduced chemotherapy‐induced cell death, as well as migration and invasion. Most of these biological modulations were rescued by AP‐2 overexpression. We observed that increased xeno‐transplant growth was mostly due to highly enhanced proliferation of the tumor cells together with reduced innate immune cell recruitment. Moreover, we showed that migration impairment was mediated, at least in part, by secreted factors. To identify the genetic programs involved in tumorigenesis, we performed whole genome microarray analysis of AP‐2α knockdown cells and observed that AP‐2α regulates specific genes involved in cell cycle, cell death, adhesion, and migration. In particular, we showed that ESDN, EREG, and CXCL2 play a major role in AP‐2 controlled migration, as ablation of any of these genes severely altered migration.—Orso, F., Penna, E., Cimino, D., Astanina, E., Maione, F., Valdembri, D., Giraudo, E., Serini, G., Sismondi, P., De Bortoli, M., Taverna, D. AP‐2α and AP‐2γ regulate tumor progression via specific genetic programs. FASEB J. 22, 2702‐2714 (2008)

Interactions between endothelial cells and HIV-1

Endothelial cells (EC) participate in inflammatory and immune reactions by producing and responding to soluble mediators. Human immunodeficiency virus (HIV)-1 profoundly alters the features of EC. In some anatomical districts, they are infected by the virus and may represent a relevant reservoir. During lymphomononuclear cell diapedesis, EC activate virus replication in crossing cells. Direct or indirect damage of EC is particularly relevant in central nervous system, where blood-brain barrier perturbation is pivotal in neuronal degeneration. The observed alterations of EC adhesive properties contribute in altered leukocyte traffic from blood to lymphoid organs and tissues and play a role in the onset of immune surveillance alteration. These alterations of EC functions are relevant for the general vasculopathy, which marks the acquired immunodeficiency syndrome, and in particular are instrumental in the pathogenesis of Kaposis sarcoma. Here we discuss the biological and molecular activation of EC in HIV-1 infection that represents the basis to understand the pathogenesis of HIV-1 associated vascular diseases.

Modulation of alpha-smooth muscle actin expression in fibroblasts by transforming growth factor-beta isoforms: an in vivo and in vitro study.

Myofibroblasts are granulation tissue fibroblasts bearing ultrastructural and biochemical features of smooth muscle cells, such as cytoplasmic microfilaments and α‐smooth muscle actin expression. They appear transiently during wound healing and more permanently during several pathologic situations such as fibrotic diseases. Transforming growth factor‐β1 has been suggested to be an important promoter of the myofibroblastic phenotype. Here we show that (1) transforming growth factor‐β2, like transforming growth factor‐β1, induces myofibroblast formation in vivo and in vitro; (2) transforming growth factor‐β3 acts as a negative regulator of the myofibroblastic phenotype in vivo but not in vitro; and (3) in vitro, the three different transforming growth factor‐β isoforms are equally able to induce α‐smooth muscle actin messenger RNA and protein expression in growing and quiescent cultured human and rat subcutaneous tissue fibroblasts. These data confirm that in vitro the behavior of the three different transforming growth factor‐β isoforms is similar, whereas in vivo transforming growth factor‐β isoforms possibly play different but complementary roles in myofibroblast modulation during wound repair.