Non-collagenous ECM proteins in blood vessel morphogenesis and cancer.

BACKGROUND: The extracellular matrix (ECM) is constituted by diverse composite structures, which determine the specific to each organ, histological architecture and provides cells with biological information, mechanical support and a scaffold for adhesion and migration. The pleiotropic effects of the ECM stem from the dynamic changes in its molecular composition and the ability to remodel in order to effectively regulate biological outcomes. Besides collagens, fibronectin and laminin are two major fiber-forming constituents of various ECM structures. SCOPE OF REVIEW: This review will focus on the properties and the biological functions of non-collagenous extracellular matrix especially on laminin and fibronectin that are currently emerging as important regulators of blood vessel formation and function in health and disease. MAJOR CONCLUSIONS: The ECM is a fundamental component of the microenvironment of blood vessels, with activities extending beyond providing a vascular scaffold; extremely versatile it directly or indirectly modulates all essential cellular functions crucial for angiogenesis, including cell adhesion, migration, proliferation, differentiation and lumen formation. Specifically, fibronectin and laminins play decisive roles in blood vessel morphogenesis both during embryonic development and in pathological conditions, such as cancer. GENERAL SIGNIFICANCE: Emerging evidence demonstrates the importance of ECM function during embryonic development, organ formation and tissue homeostasis. A wealth of data also illustrates the crucial role of the ECM in several human pathophysiological processes, including fibrosis, skeletal diseases, vascular pathologies and cancer. Notably, several ECM components have been identified as potential therapeutic targets for various diseases, including cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Mitochondrial mechanotransduction through MIEF1 coordinates the nuclear response to forces.

Tissue-scale architecture and mechanical properties instruct cell behaviour under physiological and diseased conditions, but our understanding of the underlying mechanisms remains fragmentary. Here we show that extracellular matrix stiffness, spatial confinements and applied forces, including stretching of mouse skin, regulate mitochondrial dynamics. Actomyosin tension promotes the phosphorylation of mitochondrial elongation factor 1 (MIEF1), limiting the recruitment of dynamin-related protein 1 (DRP1) at mitochondria, as well as peri-mitochondrial F-actin formation and mitochondrial fission. Strikingly, mitochondrial fission is also a general mechanotransduction mechanism. Indeed, we found that DRP1- and MIEF1/2-dependent fission is required and sufficient to regulate three transcription factors of broad relevance-YAP/TAZ, SREBP1/2 and NRF2-to control cell proliferation, lipogenesis, antioxidant metabolism, chemotherapy resistance and adipocyte differentiation in response to mechanical cues. This extends to the mouse liver, where DRP1 regulates hepatocyte proliferation and identity-hallmark YAP-dependent phenotypes. We propose that mitochondria fulfil a unifying signalling function by which the mechanical tissue microenvironment coordinates complementary cell functions.

A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1.

Cell adhesion to extracellular matrix (ECM) proteins is crucial for the structural integrity of tissues and epithelial-mesenchymal interactions mediating organ morphogenesis. Here we describe how the loss of a cytoplasmic multi-PDZ scaffolding protein, glutamate receptor interacting protein 1 (GRIP1), leads to the formation of subepidermal hemorrhagic blisters, renal agenesis, syndactyly or polydactyly and permanent fusion of eyelids (cryptophthalmos). Similar malformations are characteristic of individuals with Fraser syndrome and animal models of this human genetic disorder, such as mice carrying the blebbed mutation (bl) in the gene encoding the Fras1 ECM protein. GRIP1 can physically interact with Fras1 and is required for the localization of Fras1 to the basal side of cells. In one animal model of Fraser syndrome, the eye-blebs (eb) mouse, Grip1 is disrupted by a deletion of two coding exons. Our data indicate that GRIP1 is required for normal cell-matrix interactions during early embryonic development and that inactivation of Grip1 causes Fraser syndrome-like defects in mice.

Endothelial cell talin1 is essential for embryonic angiogenesis.

Using Tln1(fl/fl);CreER mice, we show that tamoxifen-induced inactivation of the talin1 gene throughout the embryo produces an angiogenesis phenotype that is restricted to newly forming blood vessels. The phenotype has a rapid onset in early embryos, resulting in vessel defects by 48 h and death of the embryo within 72 h. Very similar vascular defects were obtained using a Tie2-Cre endothelial cell-specific Tln1 knockout, a phenotype that was rescued by expression of a Tln1 mini-gene in endothelial cells. We show that endothelial cells, unlike most other cell types, do not express talin2, which can compensate for loss of talin1, and demonstrate for the first time that endothelial cells in vivo lacking talin1 are unable to undergo the cell spreading and flattening required to form vessels.

Regulation of lymphatic-blood vessel separation by endothelial Rac1.

Sprouting angiogenesis and lymphatic-blood vessel segregation both involve the migration of endothelial cells, but the precise migratory molecules that govern the decision of blood vascular endothelial cells to segregate into lymphatic vasculature are unknown. Here, we deleted endothelial Rac1 in mice (Tie1-Cre(+);Rac1(fl/fl)) and revealed, unexpectedly, that whereas blood vessel morphology appeared normal, lymphatic-blood vessel separation was impaired, with corresponding edema, haemorrhage and embryonic lethality. Importantly, normal levels of Rac1 were essential for directed endothelial cell migratory responses to lymphatic-inductive signals. Our studies identify Rac1 as a crucial part of the migratory machinery required for endothelial cells to separate and form lymphatic vasculature.

Vascular cell-matrix adhesion in development and cancer.

The development and homeostasis of vertebrate organisms depend on the "tree of life", in other words, the intricate network of vascular tubes composed of endothelial cells attached to the basement membrane and surrounded by perivascular cells. Although many studies have revealed the fundamental role of cytokines, growth factors and Notch signalling in vascular morphogenesis, we still lack sufficient understanding of the molecular mechanisms controlling the various steps of the angiogenic processes. Emerging data highlight that cell adhesions are key players in vascular morphogenesis. In this review, we focus on endothelial cells and we present the current state of knowledge regarding the role of cell-matrix adhesions in developmental and tumour angiogenesis, attained mainly from genetic studies and animal models.

Inactivation of AUF1 in Myeloid Cells Protects From Allergic Airway and Tumor Infiltration and Impairs the Adenosine-Induced Polarization of Pro-Angiogenic Macrophages.

The four isoforms of the RNA-binding protein hnRNPD/AUF1 have been proposed to limit the use of inflammatory mRNAs in innate immune cells. Mice engineered to lack AUF1s in all tissues are sensitive to acute inflammatory assaults; however, they also manifest complex degenerations obscuring assessment of AUF1s' roles in innate immune cells. Here, we restricted a debilitating AUF1 mutation to the mouse myeloid lineage and performed disease-oriented phenotypic analyses to assess the requirement of AUF1s in variable contexts of innate immune reactivity. Contrary to the whole-body mutants, the myeloid mutants of AUF1s did not show differences in their susceptibility to cytokine storms occurring during endotoxemia; neither in type-I cell-mediated reactions driving intestinal inflammation by chemical irritants. Instead, they were resistant to allergic airway inflammation and displayed reductions in inflammatory infiltrates and an altered T-helper balance. The ex-vivo analysis of macrophages revealed that the loss of AUF1s had a minimal effect on their proinflammatory gene expression. Moreover, AUF1s were dispensable for the classical polarization of cultured macrophages by LPS & IFNγ correlating with the unchanged response of mutant mice to systemic and intestinal inflammation. Notably, AUF1s were also dispensable for the alternative polarization of macrophages by IL4, TGFß and IL10, known to be engaged in allergic reactions. In contrast, they were required to switch proinflammatory macrophages towards a pro-angiogenic phenotype induced by adenosine receptor signals. Congruent to this, the myeloid mutants of AUF1 displayed lower levels of vascular remodeling factors in exudates from allergen exposed lungs; were unable to support the growth and inflammatory infiltration of transplanted melanoma tumors; and failed to vascularize inert grafts unless supplemented with angiogenic factors. Mechanistically, adenosine receptor signals enhanced the association of AUF1s with the Vegfa, Il12b, and Tnf mRNAs to differentially regulate and facilitate the pro-angiogenic switch. Our data collectively demonstrates that AUF1s do not act as general anti-inflammatory factors in innate immune cells but have more specialized roles in regulons allowing specific innate immune cell transitions to support tissue infiltration and remodeling processes.

Endothelial alpha3beta1-integrin represses pathological angiogenesis and sustains endothelial-VEGF.

Integrin alpha3beta1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of alpha3beta1 can affect angiogenesis either positively or negatively, either a direct in vivo role for alpha3beta1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of alpha3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where alpha3 is deleted specifically in endothelial cells (ec-alpha3-/-). Here we show that ec-alpha3-/- mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that alpha3beta1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial alpha3beta1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.

Genetic ablation of the alpha 6-integrin subunit in Tie1Cre mice enhances tumour angiogenesis.

Laminins are expressed highly in blood vessel basement membranes and have been implicated in angiogenesis. alpha6beta1- and alpha6beta4-integrins are major receptors for laminins in endothelial cells, but the precise role of endothelial alpha6-integrin in tumour angiogenesis is not clear. We show that blood vessels in human invasive ductal carcinoma of the breast have decreased expression of the alpha6-integrin-subunit when compared with normal breast tissue. These data suggest that a decrease in alpha6-integrin-subunit expression in endothelial cells is associated with tumour angiogenesis. To test whether the loss of the endothelial alpha6-integrin subunit affects tumour growth and angiogenesis, we generated alpha6fl/fl-Tie1Cre+ mice and showed that endothelial deletion of alpha6-integrin is sufficient to enhance tumour size and tumour angiogenesis in both murine B16F0 melanoma and Lewis cell lung carcinoma. Mechanistically, endothelial alpha6-integrin deficiency elevated significantly VEGF-mediated angiogenesis both in vivo and ex vivo. In particular, alpha6-integrin-deficient endothelial cells displayed increased levels of VEGF-receptor 2 (VEGFR2) and VEGF-mediated downstream ERK1/2 activation. By developing the first endothelial-specific alpha6-knockout mice, we show that the expression of the alpha6-integrin subunit in endothelial cells acts as a negative regulator of angiogenesis both in vivo and ex vivo.

The Adhesome Network: Key Components Shaping the Tumour Stroma.

Beyond the conventional perception of solid tumours as mere masses of cancer cells, advanced cancer research focuses on the complex contributions of tumour-associated host cells that are known as "tumour microenvironment" (TME). It has been long appreciated that the tumour stroma, composed mainly of blood vessels, cancer-associated fibroblasts and immune cells, together with the extracellular matrix (ECM), define the tumour architecture and influence cancer cell properties. Besides soluble cues, that mediate the crosstalk between tumour and stroma cells, cell adhesion to ECM arises as a crucial determinant in cancer progression. In this review, we discuss how adhesome, the intracellular protein network formed at cell adhesions, regulate the TME and control malignancy. The role of adhesome extends beyond the physical attachment of cells to ECM and the regulation of cytoskeletal remodelling and acts as a signalling and mechanosensing hub, orchestrating cellular responses that shape the tumour milieu.

Alphav beta3 integrin limits the contribution of neuropilin-1 to vascular endothelial growth factor-induced angiogenesis.

Both vascular endothelial growth factor receptors (VEGFR) and integrins are major regulators of VEGF-induced angiogenesis. Previous work has shown that beta3 integrin can regulate negatively VEGFR2 expression. Here we show that beta3 integrin can regulate negatively VEGF-mediated angiogenesis by limiting the interaction of the co-receptor NRP1 (neuropilin-1) with VEGFR2. In the presence of alphav beta3 integrin, NRP1 contributed minimally to VEGF-induced angiogenic processes in vivo, ex vivo, and in vitro. Conversely, when beta3 integrin expression is absent or low or its function is blocked with RGD-mimetic inhibitors, VEGF-mediated responses became NRP1-dependent. Indeed, combined inhibition of beta3 integrin and NRP1 decreased VEGF-mediated angiogenic responses further than individual inhibition of these receptors. We also show that alphav beta3 integrin can associate with NRP1 in a VEGF-dependent fashion. Our data suggest that beta3 integrin may, in part, negatively regulate VEGF signaling by sequestering NRP1 and preventing it from interacting with VEGFR2.

LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants.

Eukaryotic organisms accomplish the removal of introns to produce mature mRNAs through splicing. Nuclear and organelle splicing mechanisms are distinctively executed by spliceosome and group II intron complex, respectively. Here, we show that LEFKOTHEA, a nuclear encoded RNA-binding protein, participates in chloroplast group II intron and nuclear pre-mRNA splicing. Transiently optimized LEFKOTHEA nuclear activity is fundamental for plant growth, whereas the loss of function abruptly arrests embryogenesis. Nucleocytoplasmic partitioning and chloroplast allocation are efficiently balanced via functional motifs in LEFKOTHEA polypeptide. In the context of nuclear-chloroplast coevolution, our results provide a strong paradigm of the convergence of RNA maturation mechanisms in the nucleus and chloroplasts to coordinately regulate gene expression and effectively control plant growth.

Tumor Angiogenesis Is Differentially Regulated by Phosphorylation of Endothelial Cell Focal Adhesion Kinase Tyrosines-397 and -861.

Expression of focal adhesion kinase (FAK) in endothelial cells (EC) is essential for angiogenesis, but how FAK phosphorylation at tyrosine-(Y)397 and Y861 regulate tumor angiogenesis in vivo is unknown. Here, we show that tumor growth and angiogenesis are constitutively reduced in inducible, ECCre+;FAKY397F/Y397F -mutant mice. Conversely, ECCre+;FAKY861F/Y861F mice exhibit normal tumor growth with an initial reduction in angiogenesis that recovered in end-stage tumors. Mechanistically, FAK-Y397F ECs exhibit increased Tie2 expression, reduced Vegfr2 expression, decreased ß1 integrin activation, and disrupted downstream FAK/Src/PI3K(p55)/Akt signaling. In contrast, FAK-Y861F ECs showed decreased Vegfr2 and Tie2 expression with an enhancement in ß1 integrin activation. This corresponds with a decrease in Vegfa-stimulated response, but an increase in Vegfa+Ang2- or conditioned medium from tumor cell-stimulated cellular/angiogenic responses, mimicking responses in end-stage tumors with elevated Ang2 levels. Mechanistically, FAK-Y861F, but not FAK-Y397F ECs showed enhanced p190RhoGEF/P130Cas-dependent signaling that is required for the elevated responses to Vegfa+Ang2. This study establishes the differential requirements of EC-FAK-Y397 and EC-FAK-Y861 phosphorylation in the regulation of EC signaling and tumor angiogenesis in vivo. SIGNIFICANCE: Distinct motifs of the focal adhesion kinase differentially regulate tumor blood vessel formation and remodeling.

Efficient, inducible Cre-recombinase activation in vascular endothelium.

In recent years, gene-targeting studies in mice have elucidated many molecular mechanisms in vascular biology. However, it has been difficult to apply this approach to the study of postnatal animals because mutations affecting the vasculature are often embryonically lethal. We have therefore generated transgenic mice that express a tamoxifen-inducible form of Cre recombinase (iCreER(T2)) in vascular endothelial cells using a phage artificial chromosome (PAC) containing the Pdgfb gene (Pdgfb-iCreER mice). This allows the genetic targeting of the vascular endothelium in postnatal animals. We tested efficiency of tamoxifen-induced iCre recombinase activity with ROSA26-lacZ reporter mice and found that in newborn animals recombination could be achieved in most capillary and small vessel endothelial cells in most organs including the central nervous system. In adult animals, recombination activity was also widespread in capillary beds of skeletal muscle, heart, skin, and gut but not in the central nervous system where only a subpopulation of endothelial cells was labeled. We also tested recombination efficiency in a subcutaneous tumor model and found recombination activity in all detectable tumor blood vessels. Thus, Pdgfb-iCreER mice are a valuable research tool to manipulate endothelial cells in postnatal mice and study tumor angiogenesis.

Effects of overexpression of dimethylarginine dimethylaminohydrolase on tumor angiogenesis assessed by susceptibility magnetic resonance imaging.

Intracellular factors that regulate nitric oxide (NO) synthesis represent important targets in tumor progression. Overexpression of dimethylarginine dimethylaminohydrolase (DDAH), which metabolizes the endogenous inhibitors of NO synthesis asymmetric dimethylarginine and N-monomethyl-L-arginine, results in C6 gliomas with enhanced growth rate compared with wild type. To investigate the effects of DDAH on tumor vascular morphogenesis in vivo, we have measured the transverse relaxation rates R(2)* and R(2) in clone D27 gliomas overexpressing DDAH and C6 wild-type gliomas using intrinsic susceptibility magnetic resonance imaging (MRI), sensitive to changes in endogenous [deoxyhemoglobin], and susceptibility contrast-enhanced MRI using the intravascular blood pool contrast agent NC100150, and we compared the results with fluorescence microscopy of the tumor uptake of the perfusion marker Hoechst 33342. The baseline R(2)* was significantly faster in the D27 tumors, consistent with a greater vascular development (P < 0.02, ANOVA). There was no significant difference between the response of the two tumor types to hypercapnia (5% CO(2)/95% air), used as a probe for vascular maturation, or hyperoxia (5% CO(2)/95% O(2)), used as a probe for vascular function. NC100150 increased the R(2)* and R(2) rates of both tumor types and demonstrated a significantly larger blood volume in the D27 tumors (P < 0.02, ANOVA). This correlated with a significantly greater uptake of Hoechst 33342 in the D27 tumors compared with C6 wild-type tumors (P < 0.02, ANOVA). Despite the increased tumor blood volume, the Delta R(2)*/Delta R(2) ratio, an index of microvessel size, showed that the capillaries in the two tumor types were of a similar caliber. The data highlight the potential of susceptibility MRI-derived quantitative end points to noninvasively assess tumor angiogenesis, and in this regard, the use of intravascular blood pool contrast agents such as NC100150 appears very promising. Overexpression of DDAH results in increased neovascularization of C6 gliomas in vivo. The lack of significant difference in hypercapnic/hyperoxic response between the C6 and D27 tumors and the similar vessel caliber are also consistent with a role for DDAH in the initial stages of vasculogenesis.

Angiotensin II inhibitor facilitates epidermal wound regeneration in diabetic mice.

Tissue regeneration and wound healing are severely impaired in diabetes and are associated with poor circulation and dysfunctional blood vessels. Angiotensin II inhibitors are anti-hypertensive drugs used in clinical practice to regulate blood pressure and could affect tissue remodeling. We hypothesize that blocking angiotensin II, using Losartan, could facilitate tissue regeneration in diabetic mice. To this end, we established an experimental model of wound healing in streptozotocin-induced diabetic mice. Our data demonstrated that Losartan accelerates wound repair and normalizes wound stromal responses, having a beneficial role in wounds of diabetic individuals. Our findings highlight a potential therapeutic use of Losartan in improving wound repair in diabetic conditions.

Overexpression of dimethylarginine dimethylaminohydrolase enhances tumor hypoxia: an insight into the relationship of hypoxia and angiogenesis in vivo.

The oxygenation status of tumors derived from wild-type C6 glioma cells and clone D27 cells overexpressing dimethylarginine dimethylaminohydrolase (DDAH) was assessed in vivo using a variety of direct and indirect assays of hypoxia. Clone D27 tumors exhibit a more aggressive and better-vascularized phenotype compared to wild-type C6 gliomas. Immunohistochemical analyses using the 2-nitroimidazole hypoxia marker pimonidazole, fiber optic OxyLite measurements of tumor pO2, and localized 31P magnetic resonance spectroscopy measurements of tumor bioenergetic status and pH clearly demonstrated that the D27 tumors were more hypoxic compared to C6 wild type. In the tumor extracts, only glucose concentrations were significantly lower in the D27 tumors. Elevated Glut-1 expression, a reliable functional marker for hypoxia-inducible factor-1-mediated metabolic adaptation, was observed in the D27 tumors. Together, the data show that overexpression of DDAH results in C6 gliomas that are more hypoxic compared to wild-type tumors, and point strongly to an inverse relationship of tumor oxygenation and angiogenesis in vivo--a concept now being supported by the enhanced understanding of oxygen sensing at the molecular level.

ARF: a versatile DNA damage response ally at the crossroads of development and tumorigenesis.

Alternative reading frame (ARF) is a tumor suppressor protein that senses oncogenic and other stressogenic signals. It can trigger p53-dependent and -independent responses with cell cycle arrest and apoptosis induction being the most prominent ones. Other ARF activities, particularly p53-independent ones, that could help in understanding cancer development and provide potential therapeutic exploitation are underrated. Although ARF is generally not expressed in normal tissues, it is essential for ocular and male germ cells development. The underlying mechanism(s) in these processes, while not clearly defined, point toward a functional link between ARF, DNA damage and angiogenesis. Based on a recent study from our group demonstrating a functional interplay between ataxia-telangiectasia mutated (ATM) and ARF during carcinogenesis, we discuss the role of ARF at the crossroads of cancer and developmental processes.

FAK-heterozygous mice display enhanced tumour angiogenesis.

Genetic ablation of endothelial focal adhesion kinase (FAK) can inhibit pathological angiogenesis, suggesting that loss of endothelial FAK is sufficient to reduce neovascularization. Here we show that reduced stromal FAK expression in FAK-heterozygous mice unexpectedly enhances both B16F0 and CMT19T tumour growth and angiogenesis. We further demonstrate that cell proliferation and microvessel sprouting, but not migration, are increased in serum-stimulated FAK-heterozygous endothelial cells. FAK-heterozygous endothelial cells display an imbalance in FAK phosphorylation at pY397 and pY861 without changes in Pyk2 or Erk1/2 activity. By contrast, serum-stimulated phosphorylation of Akt is enhanced in FAK-heterozygous endothelial cells and these cells are more sensitive to Akt inhibition. Additionally, low doses of a pharmacological FAK inhibitor, although too low to affect FAK autophosphorylation in vitro, can enhance angiogenesis ex vivo and tumour growth in vivo. Our results highlight a potential novel role for FAK as a nonlinear, dose-dependent regulator of angiogenesis where heterozygous levels of FAK enhance angiogenesis.