Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A 2 /nutrin/patatin-like phospholipase domaincontaining 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44 -77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120 -210). A 25-mer peptide named P46 (aa 46 -70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition.Pigmented epithelium-derived factor (PEDF), 2 also known as SERPIN F1 and EPC1, is a 50-kDa serpin-like peptide. Although first identified in cultured pigment epithelial cells from fetal human retinas (1), we now know that liver, kidney, heart, testis, and lung tissues also express PEDF (2). PEDF influences many biological processes. It is anti-angiogenic, anti-tumorigenic, anti-inflammatory, anti-oxidative, neurotrophic, and neuroprotective, and it exhibits anti-vasopermeability properties (3-9). These diverse actions affect many cell types, including retinal cells (10), neuronal cells (11), endothelial cells (12), and hematopoietic stem cells (13). X-ray diffraction studies show that PEDF has an asymmetrical charge distribution (14). A high density of basic residues on one side of the molecule (positive) interact with heparin and glycosaminoglycans, whereas acidic residues on the opposite side (negative) interact with type-1 collagen (15)(16)(17)(18)(19)). Yet the mechanisms explaining the diverse biological activities of PEDF remain unclear.A ligand/receptor interaction at the cell membrane seemed likely, in addition to interactions within extracellular matrices, because of the diverse effects and ubiquitous expression of PEDF and the fact that most PEDF deposits remain within extracellular matrices (20). We suspected that distinct PEDF receptors elicit divergent signals to cause different biological effects. Indeed, evidence shows that PEDF binds at least two receptors: a 60-kDa receptor in ECs and an 80-kDa receptor in neuronal cells (21)(22)(23)(24). Research has identified two functional epi...

Serum response factor (SRF) is a transcription factor that controls the expression of cytoskeletal proteins and immediate early genes in different cell types. Here, we found that SRF expression is restricted to endothelial cells (ECs) of small vessels such as capillaries in the mouse embryo. EC-specific Srf deletion led to aneurysms and hemorrhages from 11.5 days of mouse development (E11.5) and lethality at E14.5. Mutant embryos presented a reduced capillary density and defects in EC migration, with fewer numbers of filopodia in tip cells and ECs showing defects in actin polymerization and intercellular junctions. We show that SRF is essential for the expression of VE-cadherin and beta-actin in ECs both in vivo and in vitro. Moreover, knockdown of SRF in ECs impaired VEGF- and FGF-induced in vitro angiogenesis. Taken together, our results demonstrate that SRF plays an important role in sprouting angiogenesis and small vessel integrity in the mouse embryo.

SUMMARYEfficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardinrelated transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

We examined the arterial phenotype of mice lacking alpha(1)-integrin (alpha(1)(-/-)) at baseline and after 4 wk of ANG II or norepinephrine (NE) administration. Arterial mechanical properties were determined in the carotid artery (CA). Integrin expression, MAPK kinases, and focal adhesion kinase (FAK) were assessed in the aorta. No change in arterial pressure was observed in alpha(1)(-/-) mice. Elastic modulus-wall stress curves were similar in alpha(1)(-/-) and alpha(1)(+/+) animals, indicating no change in arterial stiffness. The rupture pressure was lower in alpha(1)(-/-) mice, demonstrating decreased mechanical strength. Lack of alpha(1)-integrin was accompanied by an increase in beta(1)-, alpha(v)-, and alpha(5)-integrins but no change in alpha(2)-integrin. ANG II increased medial cross-sectional area of the CA in alpha(1)(+/+), but not alpha(1)(-/-), mice, whereas equivalent pressor doses of NE did not produce a significant increase in either group. In alpha(1)(+/+) mice, ANG II induced alpha(1)-integrin expression and smooth muscle cell (SMC) hypertrophy in the CA in association with increased aortic expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain and phosphorylation of ERK1/2, p38 MAPK, and FAK. ANG II did not induce SMC hypertrophy or phosphorylation of p38 MAPK and FAK in alpha(1)(-/-) mice. A functional anti-alpha(1)-integrin antibody inhibited in vitro the ANG II-induced phosphorylation of FAK and p38 MAPK. In conclusion, alpha(1)(-/-) mice exhibit a reduced mechanical strength at baseline and a lack of ANG II-induced SMC hypertrophy. These results emphasize the importance of alpha(1)beta(1)-integrin in p38 MAPK and FAK phosphorylation during vascular hypertrophy in response to ANG II.

Intermediate filaments are involved in stress-related cell mechanical properties and in plasticity via the regulation of focal adhesions (FAs) and the actomyosin network. We investigated whether vimentin regulates endothelial cells (ECs) and vascular smooth muscle cells (SMCs) and thereby influences vasomotor tone and arterial stiffness. Vimentin knockout mice (Vim−/−) exhibited increased expression of laminin, fibronectin, perlecan, collagen IV and VE-cadherin as well as von Willebrand factor deposition in the subendothelial basement membrane. Smooth muscle (SM) myosin heavy chain, α-SM actin and smoothelin were decreased in Vim−/− mice. Electron microscopy revealed a denser endothelial basement membrane and increased SM cell-matrix interactions. Integrin αv, talin and vinculin present in FAs were increased in Vim−/− mice. Phosphorylated FA kinase and its targets Src and ERK1/2 were elevated in Vim−/− mice. Knockout of vimentin, but not of synemin, resulted in increased carotid stiffness and contractility and endothelial dysfunction, independently of blood pressure and the collagen/elastin ratio. The increase in arterial stiffness in Vim−/− mice likely involves vasomotor tone and endothelial basement membrane organization changes. At the tissue level, the results show the implication of FAs both in ECs and vascular SMCs in the role of vimentin in arterial stiffening.

Rationale: Vascular smooth muscle (SM) cell phenotypic modulation plays an important role in arterial stiffening associated with aging. Serum response factor (SRF) is a major transcription factor regulating SM genes involved in maintenance of the contractile state of vascular SM cells. Objective: We investigated whether SRF and its target genes regulate intrinsic SM tone and thereby arterial stiffness. Methods and Results: The SRF gene was inactivated SM-specific knockout of SRF (SRF SMKO ) specifically in vascular SM cells by injection of tamoxifen into adult transgenic mice. Fifteen days later, arterial pressure and carotid thickness were lower in SRF SMKO than in control mice. The carotid distensibility/pressure and elastic modulus/wall stress curves showed a greater arterial elasticity in SRF SMKO without modification in collagen/elastin ratio. In SRF SMKO , vasodilation was decreased in aorta and carotid arteries, whereas a decrease in contractile response was found in mesenteric arteries. By contrast, in mice with inducible SRF overexpression, the in vitro contractile response was significantly increased in all arteries. Without endothelium, the contraction was reduced in SRF SMKO compared with control aortic rings owing to impairment of the NO pathway. Contractile components (SM-actin and myosin light chain), regulators of the contractile response (myosin light chain kinase, myosin phosphatase target subunit 1, and protein kinase C–potentiated myosin phosphatase inhibitor) and integrins were reduced in SRF SMKO . Conclusions: SRF controls vasoconstriction in mesenteric arteries via vascular SM cell phenotypic modulation linked to changes in contractile protein gene expression. SRF-related decreases in vasomotor tone and cell-matrix attachment increase arterial elasticity in large arteries.

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Synemin, a type IV intermediate filament (IF) protein, forms a bridge between IFs and cellular membranes. As an A-kinase-anchoring protein, it also provides temporal and spatial targeting of protein kinase A (PKA). However, little is known about its functional roles in either process. To better understand its functions in muscle tissue, we generated synemin-deficient (Synm 2/2 ) mice. Synm 2/2 mice displayed normal development and fertility but showed a mild degeneration and regeneration phenotype in myofibres and defects in sarcolemma membranes. Following mechanical overload, Synm 2/2 mice muscles showed a higher hypertrophic capacity with increased maximal force and fatigue resistance compared with control mice. At the molecular level, increased remodelling capacity was accompanied by decreased myostatin (also known as GDF8) and atrogin (also known as FBXO32) expression, and increased follistatin expression. Furthermore, the activity of muscle-mass control molecules (the PKA RIIa subunit, p70S6K and CREB1) was increased in mutant mice. Finally, analysis of muscle satellite cell behaviour suggested that the absence of synemin could affect the balance between self-renewal and differentiation of these cells. Taken together, our results show that synemin is necessary to maintain membrane integrity and regulates signalling molecules during muscle hypertrophy.