Impaired Cav-1 expression in SSc mesenchymal cells upregulates VEGF signaling: a link between vascular involvement and fibrosis

Fibrogenesis & Tissue Repair

Roberto Giacomelli¹  Edoardo Alesse²  Francesco Carubbi¹  Onorina Berardicurti¹  Ilenia Pantano¹  Piero Ruscitti¹  Vasiliki Liakouli¹  Francesca Zazzeroni²  Daria Capece²  Paola Di Benedetto¹  Paola Cipriani¹ 

[1] Department of Applied Clinical Sciences and Biotechnology, Rheumatology Unit, School of Medicine, University of L’Aquila, Delta 6 Building, Via dell’Ospedale, 67100 L’Aquila, Italy;Department of Applied Clinical Sciences and Biotechnology, University of L’Aquila, Coppito 2, 67100 L’Aquila, Italy

关键词: Caveolin-1;    Pericytes;    Mesenchymal stem cells;    Systemic sclerosis;   

Others  :  1121604
DOI  :  10.1186/1755-1536-7-13

 received in 2014-04-28, accepted in 2014-08-27,  发布年份 2014

【 摘 要 】

Background

Systemic sclerosis (SSc) is characterized by vascular alteration and fibrosis, the former probably leading to fibrosis via the ability of both endothelial cells and pericytes to differentiate toward myofibroblast. It is well known that vascular endothelial growth factor A (VEGF-A, hereafter referred to as VEGF) may induce a profibrotic phenotype on perivascular cells. Caveolin-1 (Cav-1) is involved in the regulation of VEGF signaling, playing a role in the transport of internalized VEGF receptor 2 (VEGFR2) toward degradation, thus decreasing VEGF signaling. In this work, we assessed the levels of Cav-1 in SSc bone marrow mesenchymal stem cells (SSc-MSCs), a pericyte surrogate, and correlate these results with VEGF signaling, focusing onpotential pathogenic pathways leading to fibrosis.

Results

We explored the VEGF signaling assessing: (1) Cav-1 expression; (2) its co-localization with VEGFR2; (3) the activity of VEGFR2, by IF, immunoprecipitation, and western blot. In SSc-MSCs, Cav-1 levels were lower when compared to healthy controls (HC)-MSCs. Furthermore, the Cav-1/VEGFR2 co-localization and the ubiquitination of VEGFR2 were impaired in SSc-MSCs, suggesting a decreased degradation of the receptor and, as a consequence, the tyrosine phosphorylation of VEGFR2 and the PI3-kinase-Akt pathways were significantly increased when compared to HC. Furthermore, an increased connective tissue growth factor (CTGF) expression was observed in SSc-MSCs. Taken together, these data suggested the upregulation of VEGF signaling in SSc-MSCs. Furthermore, after silencing Cav-1 expression in HC-MSCs, an increased CTGF expression in HC-MSCs was observed, mirroring the results obtained in SSc-MSCs, and confirming the potential role that the lack of Cav-1 may play in the persistent VEGF signaling .

Conclusions

During SSc, the lower levels of Cav-1 may contribute to the pathogenesis of fibrosis via an upregulation of the VEGF signaling in perivascular cells which are shifted to a profibrotic phenotype.

【 授权许可】

   
2014 Cipriani et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Cipriani P, Marrelli A, Liakouli V, Di Benedetto P, Giacomelli R: Cellular players in angiogenesis during the course of systemic sclerosis. Autoimmun Rev 2011, 10:641-646.
• [2]Gabrielli A, Avvedimento EV, Krieg T: Scleroderma. N Engl J Med 2009, 360:1989-2003.
• [3]Varga J, Abraham D: Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin Invest 2007, 117:557-567.
• [4]Beyer C, Distler O, Distler JH: Innovative antifibrotic therapies in systemic sclerosis. Curr Opin Rheuma 2012, 24:274-280.
• [5]Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN, Sheppard D, Chapman HA: Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A 2006, 103:13180-13185.
• [6]Wu Z, Yang L, Cai L, Zhang M, Cheng X, Yang X, Xu J: Detection of epithelial to mesenchymal transition in airways of a bleomycin induced pulmonary fibrosis model derived from an alpha-smooth muscle actin-Cre transgenic mouse. Respir Res 2007, 8:1. BioMed Central Full Text
• [7]Hashimoto N, Phan SH, Imaizumi K, Matsuo M, Nakashima H, Kawabe T, Shimokata K, Hasegawa Y: Endothelial mesenchymal transition in bleomycin-induced pulmonary fibrosis. Am J Respir Cell Mol Biol 2010, 43:161-172.
• [8]Kida Y, Duffield JS: Pivotal role of pericytes in kidney fibrosis. Clin Exp Pharmacol Physiol 2011, 38:467-473.
• [9]Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT, Bonventre JV, Valerius MT, McMahon AP, Duffield JS: Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol 2010, 176:85-97.
• [10]Duffield JS: The elusive source of myofibroblasts: problem solved? Nat Med 2012, 18:1178-1180.
• [11]Sacchetti B, Funari A, Michienzi S, Di Cesare S, Piersanti S, Saggio I, Tagliafico E, Ferrari S, Robey PG, Riminucci M, Bianco P: Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007, 19:324-336.
• [12]Tormin A, Li O, Brune JC, Walsh S, Schütz B, Ehinger M, Ditzel N, Kassem M, Scheding S: CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization. Blood 2011, 12:5067-5077.
• [13]Evensen L, Micklem DR, Blois A, Berge SV, Aarsaether N, Littlewood-Evans A, Wood J, Lorens JB: Mural cell associated VEGF is required for organotypic vessel formation. PLoS One 2009, 4:e5798.
• [14]Cai X, Lin Y, Friedrich CC, Neville C, Pomerantseva I, Sundback CA, Zhang Z, Vacanti JP, Hauschka PV, Grottkau BE: Bone marrow derived pluripotent cells are pericytes which contribute to vascularization. Stem Cell Rev 2009, 5:437-445.
• [15]Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang L, Norotte C, Teng PN, Traas J, Schugar R, Deasy BM, Badylak S, Buhring HJ, Giacobino JP, Lazzari L, Huard J, Péault BA: Perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008, 3:301-313.
• [16]Challier JC, Kacemi A, Olive G: Mixed culture of pericytes and endothelial cells from fetal microvessels of the human placenta. Cell Mol Biol 1995, 41:233-241.
• [17]Helmbold P, Nayak RC, Marsch WC, Herman IM: Isolation and in vitro characterization of human dermal microvascular pericytes. Microvasc Res 2001, 61:160-165.
• [18]Cipriani P, Marrelli A, Di B, Liakouli V, Carubbi F, Ruscitti P, Alvaro S, Pantano I, Campese AF, Grazioli P, Screpanti I, Giacomelli R: Scleroderma Mesenchymal Stem Cells display a different phenotype from healthy controls; implications for regenerative medicine. Angiogenesis 2013, 16:595-607.
• [19]Eming SA, Krieg T: Molecular mechanisms of VEGF-A action during tissue repair. J Investig Dermatol Symp Proc 2006, 11:79-86.
• [20]Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M: Growth factors and cytokines in wound healing. Wound Repair Regen 2008, 16:585-601.
• [21]Suzuma K, Naruse K, Suzuma I, Takahara N, Ueki K, Aiello LP, King GL: Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1 and phosphatidylinositol 3-kinase-akt-dependent pathways in retinal vascular cells. J Biol Chem 2000, 275:40725-40731.
• [22]Ren S, Johnson B, Kida Y, Ip C, Davidson KC, Lin SL, Kobayashi A, Lang RA, Hadjantonakis AK, Moon RT, Duffield JS: LRP-6 is co-receptor for multiple fibrogenic pathways in pericytes and myofibroblasts that are inhibited by DKK-1. Proc Natl Acad Sci U S A 2013, d110:1440-1445.
• [23]Distler O, Del Rosso A, Giacomelli R, Cipriani P, Conforti ML, Guiducci S, Gay RE, Michel BA, Brühlmann P, Müller-Ladner U, Gay S, Matucci-Cerinic M: Angiogenic and angiostatic factors in systemic sclerosis: increased levels of vascular endothelial growth factor are a feature of the earliest disease stages and are associated with the absence of fingertip ulcers. M Arthritis Res 2002, 4:R11. BioMed Central Full Text
• [24]Distler O, Distler JH, Scheid A, Acker T, Hirth A, Rethage J, Michel BA, Gay RE, Müller-Ladner U, Matucci-Cerinic M, Plate KH, Gassmann M, Gay S: Uncontrolled expression of vascular endothelial growth factor and its receptors leads to insufficient skin angiogenesis in patients with systemic sclerosis. Circ Res 2004, 95:109-116.
• [25]Harris S, Craze M, Newton J, Fisher M, Shima DT, Tozer GM, Kanthou C: VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A. Mol Cancer 2010, 31:320.
• [26]Manetti M, Guiducci S, Romano E, Bellando-Randone S, Lepri G, Bruni C, Conforti ML, Ibba-Manneschi L, Matucci-Cerinic M: Increased plasma levels of the VEGF165b splice variant are associated with the severity of nailfold capillary loss in systemic sclerosis. Ann Rheum Dis 2013, 72:1425-1427.
• [27]Harris S, Craze M, Newton J, Fisher M, Shima DT, Tozer GM, Kanthou C: Do anti-angiogenic VEGF (VEGFxxxb) isoforms exist? A cautionary tale. PLoS One 2012, 7:e35231.
• [28]Palade GE: Fine structure of blood capillaries. J Appl Physiol 1953, 24:1424-1436.
• [29]Yamada E: The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol 1955, 1:445-458.
• [30]Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG: Caveolin, a protein component of caveolae membrane coats. Cell 1992, 68:673-682.
• [31]Fra AM, Williamson E, Simons K, Parton RG: De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin. Proc Natl Acad Sci U S A 1995, 92:8655-8659.
• [32]Murata M, Peranen J, Schreiner R, Weiland F, Kurzchalia T, Simons K: VIP21/caveolin is a cholesterol-binding protein. Proc Natl Acad Sci U S A 1995, 92:10339-10343.
• [33]Labrecque L, Royal I, Surprenant DS, Patterson C, Gingras D, Béliveau R: Regulation of vascular endothelial growth factor receptor-2 activity by caveolin-1 and plasma membrane cholesterol. Mol Biol Cell 2003, 14:334-347.
• [34]Tourkina E, Richard M, Gööz P, Bonner M, Pannu J, Harley R, Bernatchez PN, Sessa WC, Silver RM, Hoffman S: Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo. Am J Physiol Lung Cell Mol Physiol 2008, 294:L843-L861.
• [35]Del Galdo F, Sotgia F, de Almeida CJ, Jasmin JF, Musick M, Lisanti MP, Jiménez SA: Decreased expression of caveolin 1 in patients with systemic sclerosis: crucial role in the pathogenesis of tissue fibrosis. Arthritis Rheum 2008, 58:2854-2865.
• [36]Haines P, Hant FN, Lafyatis R, Trojanowska M, Bujor AM: Elevated expression of cav-1 in a subset of SSc fibroblasts contributes to constitutive Alk1/Smad1 activation. J Cell Mol Med 2012, 16:2238-2246.
• [37]Krieg T, Abraham D, Lafyatis R: Fibrosis in connective tissue disease: the role of the myofibroblast and fibroblast-epithelial cell interactions. Arthritis Res Ther 2007, 9(Suppl 2):S4. BioMed Central Full Text
• [38]Dulauroy S, Di Carlo SE, Langa F, Eberl G, Peduto L: Lineage tracing and genetic ablation of ADAM12(+) perivascular cells identify a major source of profibrotic cells during acute tissue injury. Nat Med 2012, 18:1262-1270.
• [39]Scita G, Di Fiore PP: The endocytic matrix. Nature 2010, 463:464-473.
• [40]Citri A, Yarden Y: EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 2006, 7:505-516.
• [41]Mellman I: Endocytosis and molecular sorting. Annu Rev Cell Dev Biol 1996, 12:575-625.
• [42]Rink J, Ghigo E, Kalaidzidis Y, Zerial M: Rab conversion as a mechanism of progression from early to late endosomes. Cell 2005, 122:735-749.
• [43]Mosesson Y, Mills GB, Yarden Y: Derailed endocytosis: an emerging feature of cancer. Nat Rev Cancer 2008, 8:835-850.
• [44]Mukherjee S, Tessema M, Wandinger-Ness A: Vesicular trafficking of tyrosine kinase receptors and associated proteins in the regulation of signaling and vascular function. Circ Res 2006, 98:743-756.
• [45]Navarro A, Anand-Apte B, Parat MO: A role for caveolae in cell migration. FASEB J 2004, 18:1801-1811.
• [46]Shaul PW, Anderson RG: Role of plasmalemmal caveolae in signal transduction. Am J Physiol 1998, 275:L843-L851.
• [47]Liu J, Razani B, Tang S, Terman BI, Ware JA, Lisanti MP: Angiogenesis activators and inhibitors differentially regulate caveolin-1 expression and caveolae formation in vascular endothelial cells. J Biol Chem 1999, 28:15781-15785.
• [48]Liao WX, Feng L, Zhang H, Zheng J, Moore TR, Chen DB: Compartmentalizing VEGF-induced ERK2/1 signaling in placental artery endothelial cell caveolae: a paradoxical role of caveolin-1 in placental angiogenesis in vitro. Mol Endocrinol 2009, 23:1428-1444.
• [49]Wu T, Zhang B, Ye F, Xiao Z: A potential role for caveolin-1 in VEGF-induced fibronectin upregulation in mesangial cells: involvement of VEGFR2 and Src. Am J Physiol Renal Physiol 2013, 15:F820-F830.
• [50]Heerklotz H: Triton promotes domain formation in lipid raft mixtures. Biogeosciences 2002, 83:2693-2701.
• [51]Schmidt-Glenewinkel H, Reinz E, Bulashevska S, Beaudouin J, Legewie S, Alonso A, Elis R: Multiparametric image analysis reveals role of Caveolin1 in endosomal progression rather than internalization of EGFR. FEBS Lett 2012, 586:1179-1189.
• [52]Meyer C, Liu Y, Dooley S: Caveolin and TGF-β entanglements. J Cell Physiol 2013, 228:2097-2102.
• [53]Murdaca J, Treins C, Monthouel-Kartmann MN, Pontier- Bres R, Kumar S, Van Obberghen E, Giorgetti-Peraldi S: Grb10 prevents Nedd4-mediated vascular endothelial growth factor receptor-2 degradation. J Biol Chem 2004, 279:26754-26761.
• [54]Horowitz A, Seerapu HR: Regulation of VEGF signaling by membrane traffic. Cell Signal 2012, 24:1810-1820.
• [55]Le Roy C, Wrana JL: Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat Rev Mol Cell Biol 2005, 6:112-126.
• [56]Kapeller R, Chakrabarti R, Cantley L, Fay F, Corvera S: Internalization of activated platelet-derived growth factor receptorphosphatidylinositol-3 kinase complexes: potential interactions with the microtubule cytoskeleton. Mol Cell Biol 1993, 13:6052-6063.
• [57]Sigismund S, Woelk T, Puri C, Maspero E, Tacchetti C, Transidico P, Di Fiore PP, Polo S: Clathrin-independent endocytosis of ubiquitinated cargos. Proc Natl Acad Sci U S A 2005, 102:2760-2765.
• [58]Marmor MD, Yarden Y: Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases. Oncogene 2004, 23:2057-2070.
• [59]Meissner M, Reichenbach G, Stein M, Hrgovic I, Kaufmann R, Gille J: Down-regulation of vascular endothelial growth factor receptor 2 is a major molecular determinant of proteasome inhibitor-mediated antiangiogenic action in endothelial cells. Cancer Res 2009, 69:1976-1984.
• [60]Rahimi N: A role for protein ubiquitination in VEGFR-2 signalling and angiogenesis. Biochem Soc Trans 2009, 37:1189-1192.
• [61]Shi-Wen X, Leask A, Abraham D: Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine Growth Factor Rev 2008, 19:133-144.
• [62]Brigstock DR: Connective tissue growth factor (CCN2, CTGF) and organ fibrosis: lessons from transgenic animals. J Cell Commun Signal 2010, 4:1-4.
• [63]Sato S, Nagaoka T, Hasegawa M, Tamatani T, Nakanishi T, Takigawa M, Takehara K: Serum levels of connective tissue growth factor are elevated in patients with systemic sclerosis: association with extent of skin sclerosis and severity of pulmonary fibrosis. J Rheumatol 2000, 27:149-154.
• [64]Igarashi A, Nashiro K, Kikuchi K, Sato S, Ihn H, Grotendorst GR, Takehara K: Significant correlation between connective tissue growth factor gene expression and skin sclerosis in tissue sections from patients with systemic sclerosis. J Invest Dermatol 1995, 105:280-284.
• [65]Frazier K, Williams S, Kothapalli D, Klapper H, Grotendorst GR: Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol 1996, 107:404-411.
• [66]Lipson KE, Wong C, Teng Y, Spong S: CTGF is a central mediator of tissue remodeling and fibrosis and its inhibition can reverse the process of fibrosis. Fibrogenesis Tissue Repair 2012, 6(Suppl. 1):S24.
• [67]Lee CH, Shah B, Moioli EK, Mao JJ: CTGF directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model. J Clin Invest 2010, 120:3340-3349.