Structural insights into semaphorins and their receptors

Ten years ago nothing was known of the three-dimensional structure of members of the semaphorin family of cell guidance cues, nor of their major receptors, the plexins. The structural biology of this cell surface ligand–receptor system has now come of age. Detailed atomic level information is available on the architecture of semaphorin and plexin ectodomains and their recognition complexes. Similarly the structure of the plexin cytoplasmic region, and its interactions with members of the Rho family of small GTPases have been unveiled. These structural analyses, in combination with biochemical, biophysical and cellular studies, have progressed our understanding of this signalling system into the realm of molecular mechanism.     

Role of laminins in physiological and pathological angiogenesis

The interaction of endothelial cells and pericytes with their microenvironment, in particular with the basement membrane, plays a crucial role during vasculogenesis and angiogenesis. In this review, we focus on laminins, a major family of extracellular matrix molecules present in basement membranes. Laminins interact with cell surface receptors to trigger intracellular signalling that shapes cell behaviour. Each laminin exerts a distinct effect on endothelial cells and pericytes which largely depends on the adhesion receptor profile expressed on the cell surface. Moreover, proteolytic cleavage of laminins may affect their role in angiogenesis. We report in vitro and in vivo data on laminin-111, -411, -511 and -332 and their associated signalling that regulates cell behaviour and angiogenesis under normal and pathological conditions. We also discuss how tissue-specific deletion of laminin genes affects the behaviour of endothelial cells and pericytes and thus angiogenesis. Finally, we examine how coculture systems with defined laminin expression contribute to our understanding of the roles of laminins in normal and pathological vasculogenesis and angiogenesis.     

Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney

Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Their expression pattern and function outside the nervous system are unknown. Neuropilin 1 and 2 (NP-1, NP-2) are natural ligands for semaphorins 3A and 3F, respectively. NP-1 is also a co-receptor for vascular endothelial growth factor (VEGF) required for normal vascular development. We showed that VEGF is a direct chemoattractant for glomerular endothelial cells towards developing nephrons. To examine whether semaphorins could modulate VEGF endothelial cell guidance cues in the developing kidney, we studied the expression of semaphorin 3A and semaphorin 3F and their receptors NP-1 and NP-2 in the kidney during ontogeny using Northern blot analysis, in situ hybridization, Western blot analysis and immunohistochemistry. All four genes are developmentally regulated, with abundant expression during organogenesis and downregulation in the adult kidney. Semaphorin 3A and 3F are expressed by podocytes and tubules whereas their receptors NP-1 and NP-2 are localized to endothelial cells. In vitro, renal tubular epithelial cell lines (tsMPT, IRPT and MDCK) and glomerular endothelial cells express both semaphorins and their receptors, suggesting the presence of an autocrine system. The distribution of the receptors NP-1 and NP-2 in endothelial cells and developing vessels is complementary to that of the ligands in adjacent epithelial cells during kidney development. The sum of the guidance cues provided by VEGF and semaphorins 3A and 3F may be important determinants of the pattern of endothelial cell migration during kidney morphogenesis.     

Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney

Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Their expression pattern and function outside the nervous system are unknown. Neuropilin 1 and 2 (NP-1, NP-2) are natural ligands for semaphorins 3A and 3F, respectively. NP-1 is also a co-receptor for vascular endothelial growth factor (VEGF) required for normal vascular development. We showed that VEGF is a direct chemoattractant for glomerular endothelial cells towards developing nephrons. To examine whether semaphorins could modulate VEGF endothelial cell guidance cues in the developing kidney, we studied the expression of semaphorin 3A and semaphorin 3F and their receptors NP-1 and NP-2 in the kidney during ontogeny using Northern blot analysis, in situ hybridization, Western blot analysis and immunohistochemistry. All four genes are developmentally regulated, with abundant expression during organogenesis and downregulation in the adult kidney. Semaphorin 3A and 3F are expressed by podocytes and tubules whereas their receptors NP-1 and NP-2 are localized to endothelial cells. In vitro, renal tubular epithelial cell lines (tsMPT, IRPT and MDCK) and glomerular endothelial cells express both semaphorins and their receptors, suggesting the presence of an autocrine system. The distribution of the receptors NP-1 and NP-2 in endothelial cells and developing vessels is complementary to that of the ligands in adjacent epithelial cells during kidney development. The sum of the guidance cues provided by VEGF and semaphorins 3A and 3F may be important determinants of the pattern of endothelial cell migration during kidney morphogenesis.     

Adipose tissue,angiogenesis and angio-MIR under physiological and pathological conditions

Angiogenesis is a crucial process for the maintenance of normal tissue physiology and it is involved in tissue remodeling and regeneration. This process is essential for adipose tissue maintenance. The adipose tissue is composed by different cell types including stromal vascular cells as well as adipose stem cells (ASCs). In particular, ASCs are multipotent somatic stem cells that are able to differentiate and secrete several growth factors; they are recently emerging as a new cell reservoir for novel therapies and strategies in many diseases. Several studies suggest that ASCs have peculiar properties and participate in different disease-related processes such as angiogenesis. Furthermore, pathological expansion of adipose tissue brings to hypoxia, a major condition of unhealthy angiogenesis.Recent evidences have shown that microRNAs (miRNAs) play a crucial role also on ASCs as they take part in stemness maintenance, proliferation, and differentiation. It has been suggested that some miRNAs (MIR126, MIR31, MIR221 MIR222, MIR17-92 cluster, MIR30, MIR100 and MIR486) are directly involved in the angiogenic process by controlling multiple genes involved in this pathway. With the present review, we aim at providing an updated summary of the importance of adipose tissue under physiological and pathological conditions and of its relationship with neovascularization process. In particular, we report an overview of the most important miRNAs involved in angiogenesis focusing on ASCs. Hopefully the data presented will bring benefit in developing new therapeutic strategies.     

Bone marrow-derived cells are differentially involved in pathological and physiological retinal angiogenesis in mice

Purpose

Bone marrow-derived cells have been shown to play roles in angiogenesis. Although these cells have been shown to promote angiogenesis, it is not yet clear whether these cells affect all types of angiogenesis. This study investigated the involvement of bone marrow-derived cells in pathological and physiological angiogenesis in the murine retina.

Materials and methods

The oxygen-induced retinopathy (OIR) model was used as a retinal angiogenesis model in newborn mice. To block the influence of bone marrow-derived cells, the mice were irradiated with a 4-Gy dose of radiation from a ¹³⁷Cs source. Irradiation was performed in four different conditions with radio dense 2-cm thick lead disks; (1) H group, the head were covered with these discs to protect the eyes from radiation; (2) A group, all of the body was covered with these discs; (3) N group, mice were completely unshielded; (4) C group, mice were put in the irradiator but were not irradiated. On P17, the retinal areas showing pathological and physiological retinal angiogenesis were measured and compared to the retinas of nonirradiated mice.

Results

Although irradiation induced leukocyte depletion, it did not affect the number of other cell types or body weight. Retinal nonperfusion areas were significantly larger in irradiated mice than in control mice (P < 0.05), indicating that physiological angiogenesis was impaired. However, the formation of tuft-like angiogenesis processes was more prominent in the irradiated mice (P < 0.05), indicating that pathological angiogenesis was intact.

Conclusions

Bone marrow-derived cells seem to be differentially involved in the formation of physiological and pathological retinal vessels. Pathological angiogenesis in the murine retina does not require functional bone marrow-derived cells, but these cells are important for the formation of physiological vessels. Our results add a new insight into the pathology of retinal angiogenesis and bolster the hypothesis that bone marrow cells are involved in the pathology or severity of retinal angiogenic diseases.     

Endothelial cells-targeted soluble human Delta-like 4 suppresses both physiological and pathological ocular angiogenesis

Due to its essential roles in angiogenesis, Notch pathway has emerged as an attractive target for the treatment of pathologic angiogenesis. Although both activation and blockage of Notch signal can impede angiogenesis, activation of Notch signal may be more promising because it was shown that long-term Notch signal blockage resulted in vessel neoplasm. However, an in vivo deliverable Notch ligand with highly efficient Notch-activating capacity has not been developed. Among all the Notch ligands, Delta-like4(Dll4) is specifically involved in angiogenesis. In this study, we generated a novel soluble Notch ligand h D4 R, which consists of the Delta-Serrate-Lag-2 fragment of human Dll4 and an arginine-glycine-aspartate(RGD) motif targeting endothelial cells(ECs). We demonstrated that h D4 R could bind to ECs through its RGD motif and effectively triggered Notch signaling in ECs. Further, we confirmed that h D4 R could suppress angiogenesis in vitro as manifested by network formation assay and sprouting assay. More importantly, h D4 R efficiently repressed neonatal retinal angiogenesis and laser-induced choroidal neovascularization(CNV) as well in vivo. In conclusion, we have developed an in vivo deliverable Notch ligand h D4 R, which suppresses angiogenesis both in vitro and in vivo, thus providing a new approach to tackle excessive angiogenesis relevant disease such as CNV.     

From the cradle to the clinic: VEGF in developmental, physiological, and pathological angiogenesis

Formation of new blood vessels, which is fundamental in embryonic development, occurs through a combination of angiogenesis and vasculogenesis. Angiogenesis also plays a vital role postnatally, especially in reparative processes such as wound and fracture healing. Some of these events, especially in fracture healing, recapitulate processes observed in developmental angiogenesis. However, dysregulated angiogenesis is well documented to underlie a number of pathological disorders, including rheumatoid arthritis (RA). The vascular endothelial growth factor (VEGF)/VEGF receptor system is the best characterized regulator of angiogenesis. VEGF is expressed in a range of cells in response to soluble mediators (such as cytokines and growth factors), cell-bound stimuli (such as CD40 ligand), and environmental factors (such as hypoxia). As a consequence, this molecule is vital in the modulation of physiological and pathological angiogenesis. This review will focus in particular on the role played by VEGF in embryogenesis and skeletal growth, in fracture healing (in which increased angiogenesis is likely to be beneficial in promoting union), and in RA (in which excessive angiogenesis is thought to play a significant role in disease pathogenesis). In the not-too-distant future, targeting VEGF may prove to be of benefit in the treatment of diseases associated with excessive or aberrant angiogenesis, such as malignancies and RA.     

Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A?/?; Sema5B?/? mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.     

A proteome comparison between physiological angiogenesis and angiogenesis in glioblastoma

The molecular pathways involved in neovascularization of regenerating tissues and tumor angiogenesis resemble each other. However, the regulatory mechanisms of neovascularization under neoplastic circumstances are unbalanced leading to abnormal protein expression patterns resulting in the formation of defective and often abortive tumor vessels. Because gliomas are among the most vascularized tumors, we compared the protein expression profiles of proliferating vessels in glioblastoma with those in tissues in which physiological angiogenesis takes place. By using a combination of laser microdissection and LTQ Orbitrap mass spectrometry comparisons of protein profiles were made. The approach yielded 29 and 12 differentially expressed proteins for glioblastoma and endometrium blood vessels, respectively. The aberrant expression of five proteins, i.e. periostin, tenascin-C, TGF-beta induced protein, integrin alpha-V, and laminin subunit beta-2 were validated by immunohistochemistry. In addition, pathway analysis of the differentially expressed proteins was performed and significant differences in the usage of angiogenic pathways were found. We conclude that there are essential differences in protein expression profiles between tumor and normal physiological angiogenesis.     

Microparticles in physiological and in pathological conditions

Chronic diseases pose a severe burden to modern National Health Systems. Individuals nowadays have a far more extended lifespan than in the past, but healthy living was only scantily extended. As much as longer life is desirable, it is saddened by chronic diseases and organ malfunctions. One contributor to these problems was recognized to be represented by microparticles (MPs). Our purpose is to better understand MPs, to contrast their ominous threat and possible clinical importance. For this intent we correlated MPs with thrombotic pathologies, hemophilia, malaria, diabetes, cardiovascular diseases, endothelial dysfunctions, pulmonary hypertension, ischemic stroke, pre-eclampsia, rheumatologic diseases-rheumatoid arthritis, polymyositis-dermatomyositis, angiogenesis and tumor progression-cancer; we listed the possibilities of using them to improve transfusion methods, as a marker for acute allograft rejection, in stem cell transplantation, as neuronal biomarkers, to understand gender-specific susceptibility for diseases and to improve vaccination methods and we presented some methods for the detection of MPs.     

Galectin-9 in physiological and pathological conditions

We first cloned galectin-9 (Gal-9)/ecalectin as a T cell-derived eosinophil chemoattractant. Gal-9 plays a role in not only accumulation but also activation of eosinophils in experimental allergic models and human allergic patients, because Gal-9 induces eosinophil chemoattraction in vitro and in vivo and activates eosinophils in many aspects. Gal-9 requires divalent galactoside-binding activity but not the linker peptide of Gal-9 to exhibit its biological functions, and an unidentified matrix metalloproteinase is involved in the release of Gal-9. Our recent studies also showed that Gal-9 has other functions, such as cell differentiation, aggregation, adhesion, and death. Now, we and other groups are on the way of investigating the regulation and function of Gal-9 in a variety of physiological and pathological conditions. In this article, we will show the possible role of Gal-9 in physiological and pathological conditions by using our recent findings. Published in 2004.     

Galectin-9 in physiological and pathological conditions

We first cloned galectin-9 (Gal-9)/ecalectin as a T cell-derived eosinophil chemoattractant. Gal-9 plays a role in not only accumulation but also activation of eosinophils in experimental allergic models and human allergic patients, because Gal-9 induces eosinophil chemoattraction in vitro and in vivo and activates eosinophils in many aspects. Gal-9 requires divalent galactoside-binding activity but not the linker peptide of Gal-9 to exhibit its biological functions, and an unidentified matrix metalloproteinase is involved in the release of Gal-9. Our recent studies also showed that Gal-9 has other functions, such as cell differentiation, aggregation, adhesion, and death. Now, we and other groups are on the way of investigating the regulation and function of Gal-9 in a variety of physiological and pathological conditions. In this article, we will show the possible role of Gal-9 in physiological and pathological conditions by using our recent findings.     

Muscarinic receptors are involved in LMM3 tumor cells proliferation and angiogenesis

Angiogenesis is a process of new blood vessel development from pre-existing vasculature and it plays an essential role in tumor growth and metastases. Here, we investigate the expression of muscarinic acetylcholine receptors (mAchR) and their participation in tumor cell proliferation and angiogenesis ability. Saturation binding assays with the tritiated muscarinic antagonist quinuclidinyl benzilate indicate that LMM3 cells derived from a murine mammary adenocarcinoma express a single class of functional mAchR. Competition binding assays with selective muscarinic antagonists indicate a predominance of M3 receptor subtype. The muscarinic agonist carbachol (CARB) stimulates LMM3 cell proliferation in a concentration dependent manner. The maximal effect induced by 10(-9)M CARB was totally blunted by atropine and by the selective M3 and M1 antagonists, para-fluoro hexahydro sila-difenidol (pf-HHSiD) and pirenzepine, respectively. In addition, pf-HHSiD completely blocked in vivo CARB-induced neovascular formation and vascular endothelial growth factor-A in LMM3 tumor cells. We can conclude that mAchR expressed in LMM3 mammary tumor cells positively regulate proliferation and angiogenesis required for tumor progression.     

Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins.

Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.     

Tau phosphorylation: physiological and pathological consequences

The microtubule-associated protein tau, abundant in neurons, has gained notoriety due to the fact that it is deposited in cells as fibrillar lesions in numerous neurodegenerative diseases, and most notably Alzheimer's disease. Regulation of microtubule dynamics is the most well-recognized function of tau, but it is becoming increasingly evident that tau plays additional roles in the cell. The functions of tau are regulated by site-specific phosphorylation events, which if dysregulated, as they are in the disease state, result in tau dysfunction and mislocalization, which is potentially followed by tau polymerization, neuronal dysfunction and death. Given the increasing evidence that a disruption in the normal phosphorylation state of tau plays a key role in the pathogenic events that occur in Alzheimer's disease and other neurodegenerative conditions, it is of crucial importance that the protein kinases and phosphatases that regulate tau phosphorylation in vivo as well as the signaling cascades that regulate them be identified. This review focuses on recent literature pertaining to the regulation of tau phosphorylation and function in cell culture and animal model systems, and the role that a dysregulation of tau phosphorylation may play in the neuronal dysfunction and death that occur in neurodegenerative diseases that have tau pathology.