CCM1–ICAP-1 complex controls β1 integrin–dependent endothelial contractility and fibronectin remodeling

The endothelial CCM complex regulates blood vessel stability and permeability. Loss-of-function mutations in CCM genes are responsible for human cerebral cavernous malformations (CCMs), which are characterized by clusters of hemorrhagic dilated capillaries composed of endothelium lacking mural cells and altered sub-endothelial extracellular matrix (ECM). Association of the CCM1/2 complex with ICAP-1, an inhibitor of β1 integrin, prompted us to investigate whether the CCM complex interferes with integrin signaling. We demonstrate that CCM1/2 loss resulted in ICAP-1 destabilization, which increased β1 integrin activation and led to increased RhoA-dependent contractility. The resulting abnormal distribution of forces led to aberrant ECM remodeling around lesions of CCM1- and CCM2-deficient mice. ICAP-1–deficient vessels displayed similar defects. We demonstrate that a positive feedback loop between the aberrant ECM and internal cellular tension led to decreased endothelial barrier function. Our data support that up-regulation of β1 integrin activation participates in the progression of CCM lesions by destabilizing intercellular junctions through increased cell contractility and aberrant ECM remodeling.     

TGFBIp mediates lymphatic sprouting in corneal lymphangiogenesis

Corneal lymphangiogenesis plays a key role in diverse pathological conditions of the eye. Here, we demonstrate that a versatile extracellular matrix protein, transforming growth factor‐β induced protein (TGFBIp), promotes lymphatic sprouting in corneal lymphangiogenesis. TGFBIp is highly up‐regulated in inflamed mouse corneas. Immunolocalization of TGFBIp is detected in infiltrating macrophages in inflamed mouse corneas. Subconjunctival injection of liposomal clodronate can significantly reduce macrophage infiltration in inflamed mouse cornea, and decrease the expression of TGFBIp and areas of corneal lymphangiogenesis and angiogenesis after corneal suture placement. In brief, these results indicate that the up‐regulation of TGFBIp in sutured cornea correlates with macrophage infiltration. Although TGFBIp alone cannot significantly stimulate corneal lymph vessel ingrowth in vivo, it can enhance the effect of vascular endothelial growth factor‐C in promoting corneal lymphangiogenesis. The in vitro results show that TGFBIp promotes migration, tube formation and adhesion of human lymphatic endothelial cells (HLECs), but it has no effect on HLECs' proliferation. We also find that the in vitro effect of TGFBIp is mediated by the integrin α5β1‐FAK pathway. Additionally, integrin α5β1 blockade can significantly inhibit lymphatic sprouting induced by TGFBIp. Taken together, these findings reveal a new molecular mechanism of lymphangiogenesis in which the TGFBIp‐integrin pathways plays a pivotal role in lymphatic sprouting.     

Migfilin and filamin as regulators of integrin activation in endothelial cells and neutrophils

Cell adhesion and migration depend on engagement of extracellular matrix ligands by integrins. Integrin activation is dynamically regulated by interactions of various cytoplasmic proteins, such as filamin and integrin activators, talin and kindlin, with the cytoplasmic tail of the integrin β subunit. Although filamin has been suggested to be an inhibitor of integrin activation, direct functional evidence for the inhibitory role of filamin is limited. Migfilin, a filamin-binding protein enriched at cell-cell and cell-extracellular matrix contact sites, can displace filamin from β1 and β3 integrins and promote integrin activation. However, its role in activation and functions of different β integrins in human vascular cells is unknown. In this study, using flow cytometry, we demonstrate that filamin inhibits β1 and αIIbβ3 integrin activation, and migfilin can overcome its inhibitory effect. Migfilin protein is widely expressed in different adherent and circulating blood cells and can regulate integrin activation in naturally-occurring vascular cells, endothelial cells and neutrophils. Migfilin can activate β1, β2 and β3 integrins and promote integrin mediated responses while migfilin depletion impairs the spreading and migration of endothelial cells. Thus, filamin can act broadly as an inhibitor and migfilin is a promoter of integrin activation.     

Nerve Growth Factor Regulates Neurolymphatic Remodeling during Corneal Inflammation and Resolution

The cellular and physiologic mechanisms that regulate the resolution of inflammation remain poorly defined despite their widespread importance in improving inflammatory disease outcomes. We studied the resolution of two cardinal signs of inflammation–pain and swelling–by investigating molecular mechanisms that regulate neural and lymphatic vessel remodeling during the resolution of corneal inflammation. A mouse model of corneal inflammation and wound recovery was developed to study this process in vivo. Administration of nerve growth factor (NGF) increased pain sensation and inhibited neural remodeling and lymphatic vessel regression processes during wound recovery. A complementary in vivo approach, the corneal micropocket assay, revealed that NGF-laden pellets stimulated lymphangiogenesis and increased protein levels of VEGF-C. Adult human dermal lymphatic endothelial cells did not express canonical NGF receptors TrkA and p75^NTR or activate downstream MAPK- or Akt-pathway effectors in the presence of NGF, although NGF treatment increased their migratory and tubulogenesis capacities in vitro. Blockade of the VEGF-R2/R3 signaling pathway ablated NGF-mediated lymphangiogenesis in vivo. These findings suggest a hierarchical relationship with NGF functioning upstream of the VEGF family members, particularly VEGF-C, to stimulate lymphangiogenesis. Taken together, these studies show that NGF stimulates lymphangiogenesis and that NGF may act as a pathogenic factor that negatively regulates the normal neural and lymphatic vascular remodeling events that accompany wound recovery.     

Effects of basic fibroblast growth factor on human microvessel endothelial cell migration on collagen I correlates inversely with adhesion and is cell density dependent

Angiogenesis, new vessel growth from existing vessels, is critical to tissue development and healing. Much is known about the molecular and cellular elements of angiogenesis, such as the effects of growth factors and matrix molecules on proliferation and migration. However, it is not clear how these elements are coordinated to produce specific microvascular beds. To address this, the effects of basic fibroblast growth factor (bFGF) on β1 integrin-mediated adhesion relative to migration in human microvessel endothelial cells (HMVEC) was examined. Using two assays of migration that differ in the density of cells being examined, bFGF stimulated single cell migration and reduced cell migration from a confluent monolayer on collagen I. Adhesion to collagen I of HMVEC treated at low density (2−4 × 10⁴ cells/cm²) with bFGF for 22 h was reduced, while bFGF increased cell adhesion of HMVEC treated at high density (6−8 × 10⁴ cells/cm²). Adhesion of both bFGF-treated and untreated HMVEC was mediated by the β1 integrin matrix receptor. Basic FGF treatment did not significantly alter surface expression of the β1 integrin subunit. Reduction in bFGF-mediated adhesion correlated with delayed cell spreading and altered organization of β1 integrin into substrate contacts. Thus, integrin-mediated cell adhesion in microvessel endothelial cells is sensitive to regulation by a growth factor. Furthermore, the nature of the response to this signal depends on another cell regulator, cell density. In addition, modulation of cell adhesion by a growth factor may be a central regulatory feature in controlling endothelial cell migration. © 1996 Wiley-Liss, Inc.     

An essential requirement for β1 integrin in the assembly of extracellular matrix proteins within the vascular wall

β1 integrin has been shown to contribute to vascular smooth muscle cell differentiation, adhesion and mechanosensation in vitro. Here we showed that deletion of β1 integrin at the onset of smooth muscle differentiation resulted in interrupted aortic arch, aneurysms and failure to assemble extracellular matrix proteins. These defects result in lethality prior to birth. Our data indicates that β1 integrin is not required for the acquisition, but it is essential for the maintenance of the smooth muscle cell phenotype, as levels of critical smooth muscle proteins are gradually reduced in mutant mice. Furthermore, while deposition of extracellular matrix was not affected, its structure was disrupted. Interestingly, defects in extracellular matrix and vascular wall assembly, were restricted to the aortic arch and its branches, compromising the brachiocephalic and carotid arteries and to the exclusion of the descending aorta. Additional analysis of β1 integrin in the pharyngeal arch smooth muscle progenitors was performed using wnt1Cre. Neural crest cells deleted for β1 integrin were able to migrate to the pharyngeal arches and associate with endothelial lined arteries; but exhibited vascular remodeling defects and early lethality. This work demonstrates that β1 integrin is dispensable for migration and initiation of the smooth muscle differentiation program, however, it is essential for remodeling of the pharyngeal arch arteries and for the assembly of the vessel wall of their derivatives. It further establishes a critical role of β1 integrin in the protection against aneurysms that is particularly confined to the ascending aorta and its branches.     

Platelets, thrombospondin-1 and human dermal fibroblasts cooperate for stimulation of endothelial cell tubulogenesis through VEGF and PAI-1 regulation

During cutaneous wound repair, platelets, dermal fibroblasts (DF) and endothelial cells all cooperate. We have presently investigated the regulation of endothelial cell tubulogenesis by human platelet thrombospondin-1 (TSP-1), in comparison to transforming growth factor-beta1 (TGF-beta1) and total platelet lysates (PL), in a fibrin matrix cell culture system incorporating DF. TSP-1, TGF-beta1 and PL all stimulated VEGF expression in DF dose dependently at mRNA and protein level. TSP-1- and PL-treated DF supernatants significantly stimulated capillary-like structure formation (tubulogenesis) by dermal microvascular endothelial cells (HMEC-1 and HDMEC), in part via VEGF, as confirmed with neutralizing anti-VEGF antibodies. In contrast, TGF-beta1-treated DF supernatants did not induce tubulogenesis. This apparent discrepancy could be explained by the differential expression regulation in HMEC-1 of fibrinolysis and metalloproteinase mediators by TSP-1 and TGF-beta1. TSP-1 potently reduced the expression of plasminogen activator inhibitor-1 (PAI-1) (mRNA and protein), whereas TGF-beta1 enhanced it. The crucial role of PAI-1 in tubulogenesis was confirmed via the addition of active recombinant PAI-1, which abrogated tubulogenesis. In contrast, neutralizing PAI-1 antibodies enhanced tubulogenesis. Our results suggest that platelet TSP-1 released in a wound stimulates endothelial cell tubulogenesis through an upregulation of DF VEGF expression and a downregulation of endothelial cell PAI-1 expression.     

Regulation of fibronectin matrix assembly and capillary morphogenesis in endothelial cells by Rho family GTPases

Fibronectin (FN) fibrillogenesis is an essential biological process mediated by α5β1 integrin and cellular contractile forces. Assembly of a FN matrix by activated endothelial cells occurs during angiogenic blood vessel remodeling and signaling components that control this event represent attractive therapeutic targets. Here we examined the role of individual Rho GTPases in FN matrix remodeling by selectively attenuating their expression in cultured endothelial cells. Whereas pharmacological ablation of myosin-regulated contractility abrogated matrix assembly, no significant decrease was detected in the amount of FN deposited by RhoA, RhoB-, RhoC-, Rac1-, or Cdc42-depleted cells. Rather, distinct differences in fiber arrangement were observed. Most strikingly, RhoA silenced cells assembled a fine FN meshwork beneath α5β1 integrin-based fibrillar adhesions, in the absence of classical focal adhesions and actin stress fibers, indicating that α5β1 integrin translocation and FN fibril elongation can occur in low tension states such as those encountered by newly-forming vessels in tissue. In contrast, highly contractile Cdc42-deficient cells deposited FN globules and Rac-deficient cells assembled long arrays, reflecting their increased motility. We propose that regulation of FN scaffolds by Rho GTPase signaling impacts bidirectional communications and mechanical interactions between endothelial cells and their extracellular matrix during vascular morphogenesis.     

An oncogenic form of the Flt-1 kinase has a tubulogenic potential in a sinusoidal endothelial cell line

We have previously reported a constitutively activated form of the Flt-1 kinase (BCR-FLTm) molecularly engineered based on the structural backbone of the activated tyrosine kinase BCR-ABL. Here we show that it can induce not only growth stimulation but also tubulogenic differentiation of non-tubulogenic NP31 (non parenchymal) sinusoidal endothelial cells of rat liver in basement membrane matrix. Tubules formed in vitro were accompanied by fenestration structures and allowed circulation when transplanted into syngeneic animals. This biological response was not observed in other activated forms of kinases constructed in a similar fashion, which include Trk (BCR-TRK), KDR (BCR-KDR), and the parental BCR-ABL. Interestingly, formation of fine tubules was accomplished with lower but not higher expression levels of BCR-FLTm. Compared to NP cells in primary culture NP31 is deficient in expression of alpha1 integrin subunit, which was restored by expression of BCR-FLTm that had tubulogenic ability. Matrix-induced tyrosine phosphorylation of an adaptor protein Shc with recruitment of Grb-2 was observed even when tubulogenesis was nearly completed at G1 stage of the cell cycle in 2-3 weeks. Activation of matrix metalloproteinase 2 (MMP-2) and expression of urokinase type plasminogen activator (uPA) was observed with cellular invasion into matrix at the depth of 200-300 microm. Inhibitors for MAP kinase activator MEK1 and for serine proteases showed deleterious effects on the tubulogenesis. We suppose that matrix ligand-induced integrin signals cooperate with a low level of Flt-1 kinase activity to promote tubulogenic behaviors of endothelial cells in this system.     

Integrin β4 regulates SPARC protein to promote invasion

The α6β4 integrin (referred to as "β4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in β4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion. Our analysis revealed that manipulation of β4 integrin expression and signaling impacted SPARC expression and that SPARC facilitates β4-mediated invasion. Expression of β4 in β4-deficient cells reduced the expression of a specific microRNA (miR-29a) that targets SPARC and impedes invasion. In cells that express endogenous β4, miR-29a expression is low and β4 ligation facilitates the translation of SPARC through a TOR-dependent mechanism. The results obtained in this study demonstrate that β4 can regulate SPARC expression and that SPARC is an effector of β4-mediated invasion. They also highlight a potential role for specific miRNAs in executing the functions of integrins.     

Adventitial Delivery of Lentivirus-shRNA-ADAMTS-1 Reduces Venous Stenosis Formation in Arteriovenous Fistula

Hemodialysis vascular access can develop venous neointimal hyperplasia (VNH) causing stenosis. Recent clinical and experimental data has demonstrated that there is increased expression of a disintegrin and metalloproteinase thrombospondin motifs-1 (ADAMTS-1) at site of VNH. The experiments outlined in the present paper were designed to test the hypothesis that targeting of the adventitia of the outflow vein of murine arteriovenous fistula (AVF) using a small hairpin RNA that inhibits ADAMTS-1 expression (LV-shRNA-ADAMTS-1) at the time of fistula creation will decrease VNH. At early time points, ADAMTS-1 expression was significantly decreased associated with a reduction in vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase-9 (MMP-9) (LV-shRNA-ADAMTS-1 transduced vessels vs. controls). These changes in gene and protein expression resulted in favorable vascular remodeling with a significant increase in mean lumen vessel area, decrease in media/adventitia area, with a significant increase in TUNEL staining accompanied with a decrease in cellular proliferation accompanied with a reduction in CD68 staining. Collectively, these results demonstrate that ADAMTS-1 transduced vessels of the outflow vein of AVF have positive vascular remodeling.     

Smooth muscle-endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels--smooth muscle and endothelial cells--and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.     

Virally activated ras cooperates with integrin to induce tubulogenesis in sinusoidal endothelial cell lines

Four cell lines, named nonparenchymal 11 (NP11), NP26, NP31, and NP32, were established from sinusoidal endothelial cells (SECs) of rat liver. They still retained expression of receptors for vascular endothelial growth factor (VEGF), Flt-1, and kinase domain-containing receptor (KDR). NP31 and NP32 turned out to be incapable of tubulogenesis in basement membrane matrix (Matrigel), which belongs to endothelial properties, as shown by SECs in primary culture. Expression of temperature-sensitive, virally activated Ras (ts-v-Ras) restored tubulogenic behaviors back to NP31 only at permissive temperature. Matrigel induced long-lasting tyrosine phosphorylation of Shc, with recruitment of Grb-2 and microtubule-associated protein kinase (MAPK) activation in both parental NP31 and NP31 transformed by ts-v-Ras, which was blocked by anti-β1 integrin antibody. Tubulogenesis was inhibited by adenovirus-mediated expression of dominant-negative Ras in human umbilical vein endothelial cells (HUVECs). PD 098059, a selective inhibitor of MAPK kinase (MEK), nearly perfectly blocked tubulogenesis by ts-v-Ras-expressing NP31 cells at permissive temperature. Furthermore, the botulinum C3 toxin, an inhibitor for Rho, caused fragmentation of branching cords in networks formed by NP31 that expressed ts-v-Ras at permissive temperature. These data suggest that the integrin-mediated Ras signals may be necessary but are not sufficient for tubulogenesis and that an artificial expression of v-Ras might substitute for the second signal required in this system. J. Cell. Physiol. 176:223–234, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of trophoblast beta1-integrin expression by contact with endothelial cells

Background  

In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade uterine matrix, and enter the uterine vasculature. Invasive trophoblasts show increased expression of β1 integrin. Since trophoblast migration within the uterine vasculature involves trophoblast attachment to endothelial cells lining the vessel walls, this raises the possibility that cell-cell contact and/or factors released by endothelial cells could regulate trophoblast integrin expression. To test this, we used an in vitro system consisting of early gestation macaque trophoblasts co-cultured on top of uterine microvascular endothelial cells.     

Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration

Background

During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis.

Results

Protocols were developed to digitally visualize portal vessel branching and protein expression of hepatic cell lineage and extracellular matrix deposition markers in three dimensions. Samples from human prenatal livers ranging from 7 weeks + 2 days to 15½ weeks post conception as well as adult normal and acetaminophen intoxicated liver were used. The markers included cytokeratins (CK) 7 and 19, the epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex determining region Y (SRY)-box 9 (SOX9), laminin, nestin, and aquaporin 1 (AQP1). Digital three-dimensional reconstructions using CK19 as a single marker protein disclosed a fine network of CK19 positive cells in the biliary tree in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte tubulogenesis.

Conclusions

The developed protocols provide a novel and sophisticated three-dimensional visualization of vessels and protein expression in human liver during development and disease.     

Role of the α1β1 integrin complex in collagen gel contraction in vitro by fibroblasts

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the β1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-β, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the α2β1 and, to a lesser extent the α1β1 integrin complexes. The present studies were undertaken to determine the role of the α1 integrin chain, a collagen/laminin receptor, in collagen gel contration by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the α1β1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the α1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the α1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the α1β integrin complex plays a significant role in cellular interactions with interstital collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing. © 1995 Wiley-Liss, Inc.     

Caffeine suppresses the progression of human glioblastoma via cathepsin B and MAPK signaling pathway

Glioblastoma has aggressive proliferative and invasive properties. We investigated the effect of caffeine on the invasion and the anti-cancer effect in human glioblastomas. Caffeine reduced the invasion in U-87MG, GBM8401 and LN229 cells. Caffeine decreased mRNA, protein expression, and activity of cathepsin B. Besides, mRNA and protein expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) was upregulated by caffeine treatment, whereas matrix metalloproteinase-2 (MMP-2) was downregulated. The expression of Ki67, p-p38, phospforylated extracellular regulated protein kinases (p-ERK), and membranous integrin β1 and β3 was decreased by caffeine. The Rho-associated protein kinase (ROCK) inhibitor, Y27632, blocked the caffeine-mediated reduction of cathepsin B, phosphorylated focal adhesion kinase (p-FAK), and p-ERK, and invasion. Moreover, caffeine decreased the tumor size, cathepsin B and Ki67 expression in animal model. Caffeine reduced the invasion of glioma cells through ROCK-cathepsin B/FAK/ERK signaling pathway and tumor growth in orthotopic xenograft animal model, supporting the anti-cancer potential in glioma therapy.     

Id1-induced inhibition of p53 facilitates endothelial cell migration and tube formation by regulating the expression of beta1-integrin

The Id1 protein is critical for endothelial cell angiogenesis, and this function is particularly relevant to cancer development, cardiovascular disease, and wound healing. We hypothesized that Id1 enhanced migration and tubulogenesis by controlling the expression and function of p53. In this study, we examined cell migration following Id1 overexpression and silencing endothelial cells. The results showed that overexpression of Id1 enhanced cell migration and increased beta1-integrin expression, but inhibition of beta1-integrin blocked motility even in clones overexpressing Id1, suggesting that Id1 regulated motility through beta1-integrin. Further analysis revealed that p53, whose expression and distribution is regulated by Id1, was critical for cell migration, and may be involved in regulating the expression of beta1-integrin. Inhibiting p53 function using PFT-α, a functional inhibitor of p53, increased the expression of beta1-integrin and promoted cell migration even in Id1-silencing endothelial cells, demonstrating that the Id1 knockdowns induced inhibition of endothelial cell migration and the expression of beta1-integrin were controlled by p53. In addition, Id1-p53 pathway regulated the cytoskeleton formation and tubulogenesis. These results demonstrate that Id1-induced beta1-integrin expression in endothelial cells and the function of Id1 in cell migration and tubulogenesis are dependent on p53.