α-parvin is required for epidermal morphogenesis, hair follicle development and basal keratinocyte polarity

Epidermal morphogenesis and hair follicle (HF) development depend on the ability of keratinocytes to adhere to the basement membrane (BM) and migrate along the extracellular matrix. Integrins are cell-matrix receptors that control keratinocyte adhesion and migration, and are recognized as major regulators of epidermal homeostasis. How integrins regulate the behavior of keratinocytes during epidermal morphogenesis remains insufficiently understood. Here, we show that α-parvin (α-pv), a focal adhesion protein that couples integrins to actin cytoskeleton, is indispensable for epidermal morphogenesis and HF development. Inactivation of the murine α-pv gene in basal keratinocytes results in keratinocyte-BM detachment, epidermal thickening, ectopic keratinocyte proliferation and altered actin cytoskeleton polarization. In vitro, α-pv-null keratinocytes display reduced adhesion to BM matrix components, aberrant spreading and stress fibers formation, and impaired directed migration. Together, our data demonstrate that α-pv controls epidermal homeostasis by facilitating integrin-mediated adhesion and actin cytoskeleton organization in keratinocytes

Perlecan controls neurogenesis in the developing telencephalon

BACKGROUND: Perlecan is a proteoglycan expressed in the basal lamina of the neuroepithelium during development. Perlecan absence does not impair basal lamina assembly, although in the 55% of the mutants early disruptions of this lamina conducts to exencephaly, impairing brain development. The rest of perlecan-null brains complete its prenatal development, maintain basal lamina continuity interrupted by some isolated ectopias, and are microcephalic. Microcephaly consists of thinner cerebral walls and underdeveloped ganglionic eminences. We have studied the mechanisms that generate brain atrophy in telencephalic areas where basal lamina is intact. RESULTS: Brain atrophy in the absence of perlecan started in the ventral forebrain and extended to lateral and dorsal parts of the cortex in the following stages. First, the subpallial forebrain developed poorly in early perlecan-null embryos, because of a reduced cell proliferation: the number of cells in mitosis decreased since the early stages of development. This reduction resulted in a decreased tangential migration of interneurons to the cerebral cortex. Concomitant with the early hypoplasia observed in the medial ganglionic eminences, Sonic Hedgehog signal decreased in the perlecan-null floor plate basal lamina at E12.5. Second, neurogenesis in the pallial neuroepithelium was affected in perlecan deficient embryos. We found reductions of nearly 50% in the number of cells exiting the cell cycle at E12–E13. The labeling index, which was normal at this age, significantly decreased with advancing corticogenesis. Moreover, nestin(+ )or PCNA(+ )progenitors increased since E14.5, reaching up to about 150% of the proportion of PCNA(+ )cells in the wild-type at E17.5. Thus, labeling index reduction together with increased progenitor population, suggests that atrophy is the result of altered cell cycle progression in the cortical progenitors. Accordingly, less neurons populated the cortical plate and subplate of perlecan-null neocortex, as seen with the neuronal markers β-tubulin and Tbr1. CONCLUSION: As a component of the basal lamina, perlecan both maintains this structure and controls the response of the neuroepithelium to growth factors. Less mitotic cells in the early medial ganglionic eminences, and impaired cell cycle progression in the late neocortex, suggests insufficient recruitment and signaling by neurogenic morphogens, such as SHH or FGF2

Engineered microenvironments for synergistic VEGF - integrin signalling during vascularization

We have engineered polymer-based microenvironments that promote vasculogenesis both in vitro and in vivo through synergistic integrin-growth factor receptor signalling. Poly(ethyl acrylate) (PEA) triggers spontaneous organization of fibronectin (FN) into nanonetworks which provide availability of critical binding domains. Importantly, the growth factor binding (FNIII12-14) and integrin binding (FNIII9-10) regions are simultaneously available on FN fibrils assembled on PEA. This material platform promotes synergistic integrin/VEGF signalling which is highly effective for vascularization events in vitro with low concentrations of VEGF. VEGF specifically binds to FN fibrils on PEA compared to control polymers (poly(methyl acrylate), PMA) where FN remains in a globular conformation and integrin/GF binding domains are not simultaneously available. The vasculogenic response of human endothelial cells seeded on these synergistic interfaces (VEGF bound to FN assembled on PEA) was significantly improved compared to soluble administration of VEGF at higher doses. Early onset of VEGF signalling (PLCγ1 phosphorylation) and both integrin and VEGF signalling (ERK1/2 phosphorylation) were increased only when VEGF was bound to FN nanonetworks on PEA, while soluble VEGF did not influence early signalling. Experiments with mutant FN molecules with impaired integrin binding site (FN-RGE) confirmed the role of the integrin binding site of FN on the vasculogenic response via combined integrin/VEGF signalling. In vivo experiments using 3D scaffolds coated with FN and VEGF implanted in the murine fat pad demonstrated pro-vascularization signalling by enhanced formation of new tissue inside scaffold pores. PEA-driven organization of FN promotes efficient presentation of VEGF to promote vascularization in regenerative medicine applications

Perlecan-Induced Suppression of Smooth Muscle Cell Proliferation Is Mediated Through Increased Activity of the Tumor Suppressor PTEN

We were interested in the elucidation of the interaction between the heparan sulfate proteoglycan, perlecan, and PTEN in the regulation of vascular smooth muscle cell (SMC) growth. We verified serum-stimulated DNA synthesis, and Akt and FAK phosphorylation were significantly reduced in SMCs overexpressing wild-type PTEN. Our previous studies showed perlecan is a potent inhibitor of serum-stimulated SMC growth. We report in the present study, compared with SMCs plated on fibronectin, serum-stimulated SMCs plated on perlecan exhibited increased PTEN activity, decreased FAK and Akt activities, and high levels of p27, consistent with SMC growth arrest. Adenoviral-mediated overexpression of constitutively active Akt reversed perlecan-induced SMC growth arrest while morpholino antisense-mediated loss of endogenous PTEN resulted in increased growth and phosphorylation of FAK and Akt of SMCs on perlecan. Immunohistochemical and Western analyses of balloon-injured rat carotid artery tissues showed a transient increase in phosphoPTEN (inactive) after injury, correlating to high rates of neointimal cell replication; phosphoPTEN was largely limited to actively replicating SMCs. Similarly, in the developing rat aorta, we found increased PTEN activity associated with increased perlecan deposition and decreased SMC replication rates. However, significantly decreased PTEN activity was detected in aortas of perlecan-deficient mouse embryos, consistent with SMC hyperplasia observed in these animals, compared with E17.5 heterozygous controls that produce abundant amounts of perlecan at this developmental time point. Our data show PTEN is a potent endogenously produced inhibitor of SMC growth and increased PTEN activity mediates perlecan-induced suppression of SMC proliferation.Costell Rossello, M.Mercedes, [email protected]

Simultaneous boron ion-channel/growth factor receptor activation for enhanced vascularization

[EN] Boron ion is essential in metabolism and its concentration is regulated by ion-channel NaBC1. NaBC1 mutations cause corneal dystrophies such as Harboyan syndrome. Here we propose a 3D molecular model for NaBC1 and show that simultaneous stimulation of NaBC1 and vascular growth factor receptors (VEGFR) promote angiogenesis in vitro and in vivo with ultra-low concentrations of VEGF. We show Human Umbilical Vein Endothelial Cells (HUVEC) organization into tubular structures indicative of vascularization potential. Enhanced cell sprouting was found only in the presence of VEGF and boron, effect abrogated after blocking NaBC1. We demonstrate that stimulated NaBC1 promotes angiogenesis via PI3k-independent pathways and that ¿5ß1/¿vß3-integrin binding is not essential to enhanced HUVEC organization. We describe a novel vascularization mechanism that involves the crosstalk and colocalization between NaBC1/VEGFR receptors. This has important translational consequences: just by administering boron, taking advantage of endogenous VEGF, in vivo vascularization is shown in a chorioallantoic membrane assay.P.R. acknowledges support from the Ministerio de Economia, Industria y Competitividad, Gobierno de Espana (MINECO) (MAT2015-69315-C3-1-R), and European Regional Development Fund (FEDER). CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. M. S. S. acknowledges support from the European Research Council (ERC-HealInSynergy 306990) and the UK Engineering and Physical Sciences Research Council (EPSRC-EP/P001114/1). The authors are very grateful to Productos Florida farm for kindly providing chick embryos for CAM assay.Rico Tortosa, PM.; Rodrigo Navarro, A.; La Peña Del Rivero, MD.; Moulisova, V.; Costell, M.; Salmerón Sánchez, M. (2018). Simultaneous boron ion-channel/growth factor receptor activation for enhanced vascularization. Advanced Biosystems. 3(1):1-12. https://doi.org/10.1002/adbi.201800220S11231Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J., & Holash, J. (2000). Vascular-specific growth factors and blood vessel formation. Nature, 407(6801), 242-248. doi:10.1038/35025215Carmeliet, P. (2005). Angiogenesis in life, disease and medicine. Nature, 438(7070), 932-936. doi:10.1038/nature04478Moulisová, V., Gonzalez-García, C., Cantini, M., Rodrigo-Navarro, A., Weaver, J., Costell, M., … Salmerón-Sánchez, M. (2017). Engineered microenvironments for synergistic VEGF – Integrin signalling during vascularization. Biomaterials, 126, 61-74. doi:10.1016/j.biomaterials.2017.02.024Briquez, P. S., Clegg, L. E., Martino, M. M., Gabhann, F. M., & Hubbell, J. A. (2016). Design principles for therapeutic angiogenic materials. Nature Reviews Materials, 1(1). doi:10.1038/natrevmats.2015.6Hanft, J. R., Pollak, R. A., Barbul, A., Gils, C. va., Kwon, P. S., Gray, S. M., … Breen, T. J. (2008). Phase I trial on the safety of topical rhVEGF on chronic neuropathic diabetic foot ulcers. Journal of Wound Care, 17(1), 30-37. doi:10.12968/jowc.2008.17.1.27917Woo, E. J. (2012). Recombinant human bone morphogenetic protein-2: adverse events reported to the Manufacturer and User Facility Device Experience database. The Spine Journal, 12(10), 894-899. doi:10.1016/j.spinee.2012.09.052United States Food and Drug Administration Product Description Regranex https://www.fda.gov/downloads/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/UCM142821.avi (accessed: May2008).Carmeliet, P., & Jain, R. K. 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M., Kao, L., … Kurtz, I. (2009). Slc4a11Gene Disruption in Mice. Journal of Biological Chemistry, 284(39), 26882-26896. doi:10.1074/jbc.m109.008102Parker, M. D., Ourmozdi, E. P., & Tanner, M. J. A. (2001). Human BTR1, a New Bicarbonate Transporter Superfamily Member and Human AE4 from Kidney. Biochemical and Biophysical Research Communications, 282(5), 1103-1109. doi:10.1006/bbrc.2001.4692Zangi, R., & Filella, M. (2012). Transport routes of metalloids into and out of the cell: A review of the current knowledge. Chemico-Biological Interactions, 197(1), 47-57. doi:10.1016/j.cbi.2012.02.001Tanjore, H., Zeisberg, E. M., Gerami-Naini, B., & Kalluri, R. (2007). β1 integrin expression on endothelial cells is required for angiogenesis but not for vasculogenesis. Developmental Dynamics, 237(1), 75-82. doi:10.1002/dvdy.21385Gerber, H.-P., Dixit, V., & Ferrara, N. (1998). 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αv-Class integrin binding to fibronectin is solely mediated by RGD and unaffected by an RGE mutation

Fibronectin (FN) is an essential glycoprotein of the extracellular matrix; binds integrins, syndecans, collagens, and growth factors; and is assembled by cells into complex fibrillar networks. The RGD motif in FN facilitates cell binding- and fibrillogenesis through binding to α5β1 and αv-class integrins. However, whether RGD is the sole binding site for αv-class integrins is unclear. Most notably, substituting aspartate with glutamate (RGE) was shown to eliminate integrin binding in vitro, while mouse genetics revealed that FNRGE preserves αv-class integrin binding and fibrillogenesis. To address this conflict, we employed single-cell force spectroscopy, engineered cells, and RGD motif-deficient mice (Fn1ΔRGD/ΔRGD) to search for additional αv-class integrin-binding sites. Our results demonstrate that α5β1 and αv-class integrins solely recognize the FN-RGD motif and that αv-class, but not α5β1, integrins retain FN-RGE binding. Furthermore, Fn1ΔRGD/ΔRGD tissues and cells assemble abnormal and dysfunctional FNΔRGD fibrils in a syndecan-dependent manner. Our data highlight the central role of FN-RGD and the functionality of FN-RGE for αv-class integrins

Simultaneous boron ion-channel/growth factor receptor activation for enhanced vascularization

Boron ion is essential in metabolism and its concentration is regulated by ion-channel NaBC1. NaBC1 mutations cause corneal dystrophies such as Harboyan syndrome. Here we propose a 3D molecular model for NaBC1 and show that simultaneous stimulation of NaBC1 and vascular growth factor receptors (VEGFR) promote angiogenesis in vitro and in vivo with ultra-low concentrations of VEGF. We show Human Umbilical Vein Endothelial Cells (HUVEC) organization into tubular structures indicative of vascularization potential. Enhanced cell sprouting was found only in the presence of VEGF and boron, effect abrogated after blocking NaBC1. We demonstrate that stimulated NaBC1 promotes angiogenesis via Akt-independent pathways and that α5β1/αvβ3-integrin binding is not essential to enhanced HUVEC organization. We describe a novel vascularization mechanism that involves the crosstalk and colocalization between NaBC1/VEGFR receptors. This has important translational consequences: just by administering boron, taking advantage of endogenous VEGF, in vivo vascularization is shown in a chorioallantoic membrane assay

Material-driven fibronectin assembly rescues matrix defects due to mutations in collagen IV in fibroblasts

Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2+/G702D mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2+/G702D cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin β1-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2+/G702D mutation in patient-derived cells

Material-driven fibronectin assembly rescues matrix defects due to mutations in collagen IV in fibroblasts

Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2+/G702D mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2+/G702D cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin β1-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2+/G702D mutation in patient-derived cells

Profilin 1 is required for peripheral nervous system myelination

Myelination allows rapid saltatory propagation of action potentials along the axon and is an essential prerequisite for the normal functioning of the nervous system. During peripheral nervous system (PNS) development, myelin-forming Schwann cells (SCs) generate radial lamellipodia to sort and ensheath axons. This process requires controlled cytoskeletal remodeling, and we show that SC lamellipodia formation depends on the function of profilin 1 (Pfn1), an actinbinding protein involved in microfilament polymerization. Pfn1 is inhibited upon phosphorylation by ROCK, a downstream effector of the integrin linked kinase pathway. Thus, a dramatic reduction of radial lamellipodia formation is observed in SCs lacking integrinlinked kinase or treated with the Rho/ROCK activator lysophosphatidic acid. Knocking down Pfn1 expression by lentiviralmediated shRNA delivery impairs SC lamellipodia formation in vitro, suggesting a direct role for this protein in PNS myelination. Indeed,SC-specific gene ablation of Pfn1 in mice led to profound radial sorting and myelination defects, confirming a central role for this protein in PNS development. Our data identify Pfn1 as a key effector of the integrin linked kinase/Rho/ROCK pathway. This pathway, acting in parallel with integrin β1/LCK/Rac1 and their effectors critically regulates SC lamellipodia formation, radial sorting and myelination during peripheral nervous system maturation