Role of Membrane Microparticles in Angiogenesis

Hedgehog (Hh) proteins belong to a class of morphogens involved in many biological processes during embry-onic development; they are relatively silent during normal adult life although they may be recruited postnatally in re-sponse to tissue injury. Three secreted proteins have been identified: Sonic hedgehog (Shh), Desert hedgehog and Indianhedgehog. The interaction of Hh ligand with its receptor Patched-1 triggers the activation of smoothened and initiatestransduction events that lead to the regulation of transcriptional factors belonging to the Gli family. Hh pathway orches-trates both coronary development and adult coronary neovascularisation by controlling the expression of multiple pro-angiogenic genes and anti-apoptotic cytokines. Shh pathway enhances the recruitment of endothelial progenitor cells inaddition to the mechanisms described for other Hh and concurs to its myocardial protection. In cerebral ischemia, Hhmimicking molecules has been reported to limit damages caused by vessel occlusion. Besides, Shh carried by microparti-cles corrects endothelial injury through nitric oxide release. Anomalous activations of Hh pathway are implicated in vari-ous types of tumours including medulloblastoma, carcinoma of esophagus, stomach, pancreas and colon. Hh can influenceangiogenesis in both positive and negative manner and they may have implication for therapeutic strategies to treat eitherischemic or cancer diseases. INTRODUCTIONHedgehog (Hh) family proteins are morphogens widelydistributed throughout much of the animal kingdom beingfirst identified in Drosophila melanogaster [1]. In verte-brates, genome duplication has given rise to multiple Hhgenes. There are three mammalian Hh genes named Sonichedgehog (Shh), after a popular video game character, De-sert hedgehog (Dhh), after an Egyptian species of Hh (He-miechinus auritus), and Indian hedgehog (Ihh), a Hh speciesendemic in Pakistan (Hemiechinus micropus) [2-5].The core components of the Hh-family signalling path-way have been found highly conserved during the divergentevolution of insects and mammals, but Shh is the most wellcharacterized human homologue [6, 7].Hh morphogens can act as intracellular signals and areresponsible for multiple cellular fate decisions [8]. Of con-siderable importance are the roles of Hh family in the devel-opment of various embryonic tissues, including brain, spinalcord, axial skeleton, limb, lung and gut [9-12], in the vascu-larisation [13] and in the maintenance of adult tissue homeo-stasis, tissue repair during chronic persistent inflammation,and carcinogenesis [14-19].The present review summarizes the main molecularmechanisms of the Hh signalling as well as the consequencesof Hh pathway activation in pathologies such as ischemicand cancer diseases. Finally, we propose helpful indicationsfor the design of effective strategies in using molecules act-ing on Hh pathway. *Address correspondence to this author at the CNRS UMR, 6214, Facultéde Médecine, Rue Haute de Reculée, Angers, F-49045 France; Tel: +33 241 73 58 57; Fax: +33 2 41 73 58 95;E-mail: [email protected] SIGNALLING PATHWAYDespite extensive studies addressing the regulation andfunction of the pathway in different organs, the exact mecha-nisms by which Hh signals are transmitted and how theyelicit diverse activities in a cell-specific manner remain ob-scure.Newly synthesized Hh proteins undergo a series of post-translational processing reactions within the secretory path-way that result in the formation and cell surface presentationof the active species in signalling (Fig. 1). Although ele-ments of the reaction mechanisms employed are also repre-sented in the metabolism of other proteins, Hh family mem-bers are the only examples of signalling proteins known tobe covalently modified by cholesterol [20].Hh is translated into an approximately 47 kDa precursorprotein and, during their maturation, undergoes two lipo-philic modifications. Following cleavage of an amino-ter-minal signal sequence upon entry into the secretory pathway,the Hh protein undergoes an autocatalytic processing reac-tion that involves intramolecular cleavage between Gly-Cysresidues that form part of an absolutely conserved Gly-Cys-Phe tripeptide [12, 21-23].A 19 kDa amino-terminal product (termed Hh-Np) isresponsible for the signalling activity of Hh, whereas a 26kDa carboxy-terminal product functions as a cholesteroltransferase, covalently modifying the N-terminal product onits terminal amino acid [24, 25]. The amino-terminal productof this cleavage receives a covalent cholesteryl adduct [24].Besides, the N-terminal product of Hh proteins is subjectedto a second modification that is catalyzed by an acyltrans-ferase encoded by Skinny hedgehog (ski). This additionalmodifying adduct is a fatty acid, usually palmitate, and istel-00441925,version1-17Dec2009 2 Current Signal Transduction Therapy, 2009, Vol. 4, No. 1Porro et al. found in an amide linkage with the amino-terminal cysteinethat is exposed by signal sequence cleavage. Because thisCys residue is the first of a pentapeptide, which is widelyconserved among species, there is a possibility that theseresidues and others nearby may constitute an important de-terminant for the palmitoylation reaction [20, 26]. Thesehydrophobic modifications have profound effects on theproperties of Hh proteins, because they promote retention ofHh in the plasma membrane and, paradoxically, play a cru-cial role in the regulation of the range of Hh signalling in atissue.Conflicting reports have been published on the role ofcholesterol moiety in Hh signalling. The cholesterol modifi-cation anchors Hh-Np to the membrane of the producingcells and therefore originally Hh-Np is considered mainlyresponsible for short-range signalling. However, subsequentstudies have shown that Hh-Np is essential for the activity ofHh signalling, especially the long-range effects far from theproducing cells [21, 27-29]. The palmitoylation is essentialto both the activity of the Hh protein and the signal range [5,30, 31].Since most Hh proteins are anchored on the cell mem-brane attributing to lipid modification, various mechanismshave been proposed for Hh long-range effects, three ofwhich are active diffusion through extracellular matrix, indi-rect transmission through secondary signal cascade and cy-Fig. (1). Schematic diagram of Hh protein biogenesis and signalling pathway. Hh protein is synthesized as a 47 KDa precursor protein;its auto processing generate N-terminal domain to yield a 19KDa product, dually lipidate that mediates signalling. C-terminus receives cho-lesterol by 26 KDa C-terminal product of auto cleavage and N-terminus receives palmitic acid by Ski. Hh protein is then trafficked to cellsurface and released by aid of Disp and other cellular activities such Megalin and Glipicans. Hh signalling regulates balance of its transcrip-tional factor target, Gli, from the repressor form to activator form. On receiving cells, in absence of Hh, Ptc blocks Smo transduction func-tion. This inhibition favours the cytoplasmic retention and inactivation of Gli. Repressed Smo induces the assembly of complex of full-lengthGli and SUFU and, also, of the cytoplasmic Gli degradation complex, in which Gli is phosphorylated initially by CKI and then by GSK andPKA. Hyperphosphorylated Gli and its subsequent polyubiquitination induce its processing by proteasome. Released truncated Gli enters thenucleus and represses the transcription of target genes. Hh-binding to complex of Ptc and CDO/BOC but also with other proteins, such Hipand Gas1, derepresses the activity of Smo, allowing the activation of STK36 serine/threonine kinase. STK36 inhibits the formation of Glidegradation complex and the phosphorylation of SUFU, promoting stabilization of full-length Gli and its accumulation into nucleus, where itcan bind DNA and regulate the expression of its target genes.tel-00441925,version1-17Dec2009 Sonic Hedgehog Pathway as a TargetCurrent Signal Transduction Therapy, 2009, Vol. 4, No. 1 3 tonemes through cellular extensions [32-35]. Also, Zeng etal. [35] have shown the presence of two forms of solubleShh-Np, far from producing source, namely monomer andmultimer. In addition, they reported that the multimeric formcannot be observed if cholesterol modification is disrupted,leading to the hypothesis that Shh-Np embeds the lipid moie-ties in the hydrophobic surface to form the multimers andthereby diffuse freely to the distal compartment. Furtherstudies have demonstrated that protein-protein and protein-lipid interactions are required for Shh-Np multimer forma-tion [36, 37]. Another suggestion based in the ability of Hhto remain anchored on the plasma membrane has been re-cently reported. Martínez and co-workers [38] have shownthat long-range Hh signal can be mediated also when Shh iscarried by microparticles (MPs), small membrane fragmentsshed from blebbing plasma membrane of various cell types,such as platelets, T and B cells, monocytes, and endothelialcells during activation by agonists, shear stress or apoptosis[39]. MPs harbouring the morphogen Shh (MPsShh+), gener-ated from engineering human T lymphocytes undergoing acti-vation/apoptosis or from plasma from diabetic patients, arefunctionally active and induce intracellular responses in re-ceiving cells probably due to ligand/receptor interaction [38].Because of lipid modifications, specific cellular activitiesare required in signal-generating and in signal-receiving cells(Fig. 1). These include Dispatched (Disp) and Exotosin(EXT, homologous to Drosophila tout-velu, Ttv). Dispatchedis a 12-pass transmembrane-domain protein. Five adjacenttransmembrane segments compose a sterol-sensing domainrequired for Hh release and are also representative of othermembers of proteins that include Patched (Ptc-1), a compo-nent of the Hh receptor [40-42]. By contrast, EXT regulatessynthesis of proteoglycans and functions to allow traffickingof Hh. Hh is also affected by the glycoprotein Hh-interactingprotein (Hip) to which Hh binds with high affinity. Hip isinduced in receiving cells and acts in parallel to Ptc-1 as anattenuator of the Hh response. Hip is also a target gene in Hhsignalling in a negative feedback regulating manner [43-46].Moreover, Hh trafficking potentially involves interactionwith other proteins such glypicans, megalin and growth ar-rest-specific gene 1 (Gas1). Glypicans such Dally and Dally-like proteins (Dlp) are substrate of Ttv and transfer Hh alongthe cell membrane [47]. Megalin and Gas1 are proteins thatcan bind Shh and can also modulate its activity [48]. Addi-tional proteins function to receive the Hh signal: Patched,CDO (cell-adhesion-molecule related/down-regulated byoncogenes, also known as CDON), BOC (bioregional celladhesion molecule-related/down-regulated by oncogenes(Cdon) binding protein) and Smoothened (Smo) [49].Unusually for a signal receptor, Ptc-1 is not activated inresponse to ligand binding but rather is suppressed by itsinteraction with Hh. Two Ptc genes exist in vertebrates, Ptc1and Ptc2. These genes encode for 12-pass transmembranereceptors with two extracellular hydrophilic loops that medi-ate Hh binding [50, 51]. Smo is a 7-transembrane-domainprotein homologous to G-protein-coupled receptors. It doesnot bind Hh directly but acts a focal point of Hh signal trans-duction [14, 15, 17, 19, 52-56]. In its unbound state, Ptc-1represses the activity of Smo signal transducer, which is nec-essary for the transduction of the Hh signalling across theplasma membrane and into the cell. Therefore Hh activatesSmo by binding to inactivating Ptc-1. Hh may induce Smo tochange its conformation or to oligomerize. Recent findingsprovide evidence for constitutive Smo dimers or multidimersthat adopts different intracellular conformation in response toHh. It is interesting to note that Smo is more fully phos-phorylated, it localizes more predominantly to the plasmamembrane as opposed to internal vesicles and it is present athigher overall levels [17, 57].Cyclopamine, which is isolated from the corn lily Vera-trum californicum, and synthetic small molecules includingCUR61414 and SANT1-4 have been identified as modulat-ing Smo activity. It has been proposed that they act at levelof the hydrophobic domain delineated by 7-putative trans-membrane domains of Smo. Another class of molecules in-cluding SAG derived from a chlorothiophen moiety acts asSmo agonists when applied to Hh responding cells [58-61].Also, it has been showed that vitamin D3 and pro-vitaminD3 bind to Smo with high affinity and inhibit its activity[62]. Moreover, sterol synthesis inhibitors reduce Shh targetgene transcription and block Shh pathway-dependent prolif-eration probably acting on the pathway upstream or at thelevel of Smo [63].Once Shh binds to Ptc-1, the activity of Smo is no longerinhibited and multimolecular network transduces Hh signalthrough activation and nuclear translocation of members ofthe glioma-associated oncogene (Gli) family of transcriptionfactors (Gli1, Gli2 and Gli3). The process by which Gli isregulated consists in post-transcriptional transition and bal-ance of Gli from the repressor form to activator form. TheGli proteins bind DNA in a sequence-specific manner throughthe last three fingers of 5-zinc-finger domain [64]. They havespecialized functions and distinct temporal and spatial ex-pression patterns. Gli1 can only act as an activator of tran-scription, whereas Gli2 and Gli3 can be processed to func-tion as transcriptional activators or repressors [65, 66].Gli proteins activity is controlled by two different com-plexes (Fig. 1). The first is a multicomponent cytoplasmiccomplex which includes the kinesin-like proteins KIF7 andKIF27 [67], the serine/threonine kinase Fused (FU) [68],casein kinase I (CKI), glycogen synthase kinase-3ß (GSK3ß)and protein kinase A (PKA). Inactivated Smo induces theformation of the cytoplasmic Gli degradation complex, inwhich Gli members are phosphorylated initially by CKI, andsubsequently by GSK3ß and PKA [69, 70]. Hyperphoshory-lated Gli confers binding to ßTRCP, an ubiquitin F-box pro-tein [71-73]. Subsequently polyubiquitinated Gli is processedby proteasome machinery to release its intact N-terminalmoiety that enters the nucleus and represses the transcriptionof target genes. The second complex composed of full-lengthGli and the suppressor of Fused (SUFU) [74] results in thecytoplasmic retention of Gli and then in its inactivation.With regard to the second complex, Hh-binding to Ptc-1receptor releases the Smo signal transducer from Ptc-1-depen-dent suppression. Then, Smo activates STK36 serine/threo-nine kinase to inhibit the assembly of Gli degradation com-plex allowing the stabilization of full-length Gli [75]. Acti-vated STK36 also phosphorylates SUFU to promote accumu-tel-00441925,version1-17Dec2009 4 Current Signal Transduction Therapy, 2009, Vol. 4, No. 1Porro et al. lation of full-length Gli into nucleus [76], where it can bindDNA and regulate the expression of its target genes.To date, only a small number of Hh target genes havebeen characterized in detail. Ptc-1 gene is a target of its ownrepression, and its transcription is elevated upon stimulationwith Hh. This up-regulation may antagonize further Hhstimulation, or may play a direct role in cell cycle control asPtc-1 is reported to interact with cyclin B1 (Barnes et al.,2001). Gli1 and Hip are transcriptional targets of Hh signal-ling pathway implicated in the negative feedback mechanismof Hh cascade [77, 78]. Cell cycle regulators like cyclin D1and D2, c-Myc, N-Myc and L-Myc, as well as Forkhead-boxtranscription factors, are reported to be upregulated by theHh signalling [14, 17, 52, 67, 79, 80]. Other targets includeHNF-3b [27] in the developing neural tube, SWiP-1 [81] andSFRP-2 [82] in somitic mesoderm, and angiopoietin 2 (Ang-2) in developing vasculature [83]. Other tissue-specific tar-gets include members of the bone-marrow protein family ingastrointestinal tract [84], PAX family in motor neurons dif-ferentiation [85], SOX family in spinal cord [86], and TBXfamily in developing hind limb and forelimb [87].Complexity of Hh cascade is also provided by its cross-talk with other pathways. Additional proteins have beenidentified as components of mammalian Hh signalling suchas PI3K, Akt, PKC , MEK-1, IFT88, IFT172, SIL, Kif3a,MIM/BEG4, -arrestin2, and Rab23 [88-94]. For example,Smo can activate non canonical signals, like PI3K and Akt.At the same times, Hh pathway is subjected to regulation byother signal cascades, like PKC /MEK-1 pathway. Hh sig-nalling can be modulated by parallel activation of additionalintracellular signalling routes, such as those triggered by epi-dermal growth factor or fibroblast growth factor [95, 96].Recently, we reported that MPsShh+trigger changes in theexpression and phosphorylation of enzymes related to thenitric oxide (NO) pathway. Shh carried by MPs promote, inendothelial cells, endothelial NO-synthase (eNOS) expres-sion and module its activity as revealed by increasing ofphosphorylation of activator site (Ser-1177) by a cyclopa-mine-sensitive mechanism [97].The Hh family of proteins is powerful morphogen medi-ating embryonic development but several recent studies re-veal its implication during adult life such in neovascularisa-tion through an upregulation of multiple families of angio-genic growth factors (VEGF, Ang) [83].EFFECTS OF Shh IN NEOVASCULARISATIONPostnatal neovascularisation, including both angiogenesisand vasculogenesis, is regulated by a complex interplayamong various growth factors, circulating bone marrow-deri-ved precursor/stem cells, endothelial and inflammatory cells[98, 99]. Whereas angiogenesis is defined as the formation ofnew blood vessels by way of sprouting of pre-existing ma-ture endothelial cells [98], vasculogenesis is referred to theformation of the earliest vascular network via the differentia-tion of endothelial progenitor cells into endothelial cells[100].In established blood vessels in mature organisms, endo-thelial cells remain in a quiescent, non-proliferate state untilstimulation of angiogenesis occurs. The formation of newvessels can be considered as the result of several processes:(i) dissolution of the matrix underlying the endothelial celllayer; (ii) migration, adhesion and proliferation of endothe-lial cells; (iii) formation of a new three-dimensional tube,which then lengthens from its tip as circulation is re-established; and (iv), in larger vessels, vascular smooth mus-cle cells also migrate and adhere to the newly deposited ma-trix of the native vessel [101].Angiogenic growth factors induce, promote and/or inter-fere with all these steps of angiogenesis [102-104]. The vari-ety of growth factors plays significant role in cell prolifera-tion, maturation and differentiation leading to the formationof mature blood vessels. These factors act as signallingmolecules between cells, and bind to specific receptors onthe surface of their target cells. The best known growth fac-tors with proven angiogenic potency are the family of fibro-blast growth factors (FGF) and VEGF [101], insuline-likegrowth factor-1 (IGF1), Ang, and hepatocyte growth factor(HGF) [105].In 2001, Pola et al [83]. described for the first time theinvolvement of the Hh family of morphogens in angiogene-sis. There are several features worthy of consideration in Hhsignalling in angiogenesis. First, the physiological functionsof Hh signalling are broader than other the so called specificangiogenesis factors, such as VEGF or Ang. Secondly, theirrole in embryogenesis is temporo-spatially determined by thedevelopmental stage and context [106]. Thus, Hh signallingmight be better considered as a coordinator of the cross-talkbetween angiogenesis and other developmental processessuch as neurogenesis. Therefore, due to its pleiotropic func-tion, targeting Hh signalling might affect both angiogenesisand morphogenesis. Thus, care must be taken when usingsuch strategy.Shh is involved in de novo vascularisation of certain em-bryonic tissues. Several studies have reported that Shh sig-nalling derived from the notochord stimulates the formationof the aorta in the zebrafish embryo [107]. Vokes et al. [108]described essential roles for Shh signalling in dorsal aortaformation in avian and mouse embryos. Indeed, Shh is ex-pressed in the avian endoderm and formation of the dorsalaorta is significantly inhibited in the endodermless or cyclo-pamine-treated avian embryo, as well as, in Smo-deficientmouse embryos in first embryonic days.Besides, Shh signalling stimulates angiogenesis in vari-ous embryonic tissues at late developmental stages includingits release from epicardial layer of the heart in mouse em-bryos [109]. Shh expression is triggered by FGF and result-ing in upregulation of the expression of VEGF and Ang-2 inthe myocardium. In mouse embryos, transgenic mice revealthat overexpression of Shh leads to hypervascularisation ofthe neuroectoderm [110]. In contrast, mutant zebrafishs withdeficient Hh signalling have defects in circulation and vascu-larisation including the formation of a single axial vesselwith no arterial markers [107, 111]. The latter is implicatedin the development of coronary vessels in the subepicardialspace and within the myocardium.Shh signalling also plays a role in the developing lungvasculature. Indeed, Van Tuyl et al. [112] reported that thetel-00441925,version1-17Dec2009 Sonic Hedgehog Pathway as a TargetCurrent Signal Transduction Therapy, 2009, Vol. 4, No. 1 5 expression of Ang-1 and its receptor Tie-2 are downregu-lated in the lungs of Shh null embryos. In this study theauthors advance the hypothesis that defective angiogenesisresults in impaired airway branching [112] and thereforemice lacking Shh function exhibit poor vascularisation ofdeveloping lung [113]. Of note is the fact that Shh can actsynergistically with other growth factors such as FGF9 inpulmonary capillary network [114]. In this context, themechanism involves mesenchymal expression of VEGF butnot Ang-1.Shh AND PATHOLOGIES ASSOCIATED WITHISCHEMIAAmong Hh-regulated processes in adults, revasculariza-tion of ischemic tissue is of the outmost clinical importance[115]. Ischemic injury results in deprivation of oxygen andnutrients, and then the growth and viability of cells is re-duced. The Hh-dependent revascularization probably reflectsa physiological response to ischemic stress (hypoxia or in-flammation). To date, the studies are conduced to understandShh potentialities in the rescue of tissues that can be sub-jected to ischemic insults. Reports documents positive ef-fects of this morphogen in different tissue such as myocar-dium [116], cornea and hindlimb [13], kidney [117] andbrain [53, 118].With regard to wound healing, Asai et al. [119] reportedthat endogenous Shh signalling is activated in a mousewound model. Thus, the transfection of naked Shh-DNApromotes wound healing via vasculogenesis, i.e. recruitmentof endothelial progenitor cells from the bone marrow. How-ever, no clear evidence is shown for the involvement of acti-vated endogenous Shh signalling in the wound healing proc-ess because pharmacological inhibitors of the downstreamsignalling pathway have not been used. Nevertheless, thesedata point to the involvement of Shh signalling in angio-genesis and vasculogenesis in response to skin injury.It has been shown that administration of recombinant Shhprotein induces angiogenesis in ischemic limbs and cornea ofadult mice [83], indicating the involvement of Hh in postna-tal vascular development. The same group has reported theinduction of nerve vessels and restoration of nerve functionin rat diabetic neuropathy after systemic injection of recom-binant Shh protein, suggesting a therapeutic angiogenic po-tential of exogenous Shh. In these cases, VEGF expressionwas upregulated by Shh signalling in the cornea stromal cellsand ‘‘nerve fibroblasts’’, respectively [120].It is of interest to consider whether endogenous Shh sig-nalling in postnatal vascular development is a physiologicaland/or repair process. Shh expression is upregulated in re-generating muscle in mouse ischemic legs [13]. Thus, ad-ministration of a Shh-neutralising antibody inhibited angio-genesis and VEGF upregulation in this model, implying arole for endogenous Shh signalling in ischemic angiogenesis.Similar findings have been reported in a study in which reti-nal angiogenesis is associated with upregulation of Shh sig-nalling and VEGF by a mechanisms sensitive to cyclopa-mine [121].A field of recent interest is the potential of modulatingShh signalling as a therapy for myocardial ischemia. Wehave provided data that Shh carried by MPs. are able to re-store endothelial dysfunction in a model of mice coronaryischemia by a mechanism sensitive to cyclopamine.(see be-low) [97].Conditional activation of Gli-2, which is downstream ofShh activation, resulted in increased coronary vessel densitywithin the myocardium along with upregulation of VEGF inmouse adult heart [109]. Furthermore, Shh gene therapy re-stores myocardial function in a mouse model of acute orchronic myocardial infarction. Indeed, transfection of DNAencoding human Shh not only promotes neovascularisationvia, at least in part, recruitment of endothelial progenitorcells, but also reduces cardiac fibrosis and apoptosis. Severalangiogenic factors, such as VEGF and Ang, are upregulatedby Shh in the cardiac fibroblasts [116]. Altogether, these datasupports the view that Shh signalling during ischemia resultsin upregulation of VEGF, thereby promoting angiogenesis.With regard to cerebral ischemia, a positive role for Hh-mimicking molecules has also been reported to limit thedamage caused by artificial vessel occlusion in rats [53,118].One can advanced two theories to explain the effects ofmorphogens in angiogenesis (Fig. 2). Secreted Hh ligandscould act directly on the endothelial cells to stimulate prolif-eration and vessel formation. Alternatively, the more com-monly accepted theory is that Hh ligands act on support cells(pericytes and smooth muscle cells), the nature of which isstill not fully elucidated, which produce vascular growthfactors, such as VEGF to promote angiogenesis [122].Supporting the first assumption, Geng and co-workers[123] suggest a direct interaction between Hh and endothe-lial cells. They have shown, in an in vitro single cell line thatexogenous treatment with Shh stimulates proliferation ofendothelial cells and blockade of the Hh pathway reducesthis response. Besides, the group of Hochman [124] reportsthat endothelial cells derived from the mouse embryo yolksac, as well as, embryonic mouse fibroblasts respond directlyto Hh by transcriptional activation of multiple genes codingfor proteins involved in migration and angiogenesis. Theyfurther point out that Hh signalling enhances in vitro woundhealing via neuropilin-flavomonooxygenase pathway. In linewith the first theory, Shh mediates in vitro migration andcapillary formation of mature endothelial cells and putativeendothelial progenitor cells from the bone marrow [119, 124,125]. Altogether, these studies underscore endothelial cellsas direct targets of Shh.On the other hand, many reports have also shown indirectactions of Shh-mediated angiogenesis. Shh-dependent aortaformation in zebrafish, reported by Lawson et al. [107], isalso mediated by expression of VEGF in the mesoderm. Shhtreatment, in vitro, upregulates angiogenic genes, such asVEGF and Ang in NIH3T3 embryonic fibroblasts, as well as,in adult dermal fibroblasts [83, 126]. Thus, Shh promotes itsaction secondary to the release of proangiogenic factors.Altogether, these findings support a dual mechanism ofShh-induced angiogenesis, namely, direct and indirect. Eventhough Shh directly interacts with endothelial cells, in vitro,this finding does not rule out the indirect interaction. It ispossible that Hh peptides affect both the stroma and the en-dothelium.tel-00441925,version1-17Dec2009 6 Current Signal Transduction Therapy, 2009, Vol. 4, No. 1Porro et al. The exact Shh pathway involved in the reduction of ische-mia is not fully understood, even if some cross-link betweenHh pathway and proangiogenic molecules have been re-ported. Growing evidence reveals that among the pro-angiogenic factors that could be possible target of Shh path-way to reduce ischemia, hypoxia-inducible factors (HIF)[127], insulin-like growth factor (IGF)-2 [128], nitric oxide(NO) [129] and reactive oxygen species (ROS) [130] are themost representative.It is important to note that the most powerful activator ofangiogenesis is hypoxia. Under hypoxic conditions, hypoxia-induced mitogenic factor, promotes proliferation and migra-tion of endothelial cells [105] and low levels of reactiveoxygen species (ROS) (which are produced in response togrowth factor, tissue ischemia/hypoxia or ischemic precondi-tioning) function as signalling molecules to mediate endothe-lial cell migration [131-133] and may contribute to angio-genesis in vivo [134, 135].One of the main targets of hypoxia is HIFs. HIFs aretranscription factors that activate pathways with the ability toincrease the oxygen supply and promote adaptive responsesto stress. Among the multiple targets of HIF genes areVEGF, erythropoietin, Ang, placental growth factor, andplatelet-derived growth factor, indicating that HIF-1 func-tions as a master regulator of angiogenesis in ischemic tis-sues [136]. These angiogenic factors recruit subsets ofproangiogenic hematopoietic cells along with endothelialprogenitor cells. These, in turn, may also release new angio-genic factors contributing to the repair by paracrine effects.Indeed, hypoxic preconditioning of endothelial progenitorcells can increase their ability to repair ischemic limbsthrough the activation of the angiogenic program. [137].There are some reports showing that HIF-1 and Shh withother factors can act in synergy to protect tissue againstischemic insults. Indeed in embryo rat heart, transient ische-mia induces expression of HIF-1 , Shh, IGF-2, and VEGFgenes, suggesting that these proteins may play a role in car-dioprotective effect especially in cardiomyoblast. HIF-1regulates some proteins adapted to hypoxia, including IGF-2and VEGF; IGF-2 improves growth and proliferation; andShh improves myogenesis in synergy with IGF .The expo-sure of cardiomyoblast cells to HIF-1 , and Shh inhibitorsrespectively, resulted in the cross-inhibition of each of thepathway. Moreover a complex cross-talk between these pro-teins has been proposed and could have important implica-tions in the understanding of proliferation and angiogenesisfollowing ischemia [127]. These results are also supportedby a recent study in which the authors have highlighted therole of IGF-2 in Shh signalling and angiogenesis. They ad-vance the hypothesis that IGF-2 may mediate the angiogeniceffects of Shh, providing a critical link between Shh andVEGF [128].The involvement of NO in ischemia-induced angiogene-sis is supported by the studies performed by Luque Contrerasand co-workers [129], which show that (i) NO levels areincreased in the ischemic limb; (ii) pharmacological inhibi-tion or gene disruption of endothelial NO-synthase decreasesNO levels and inhibits ischemia-induced angiogenesis; (iii)supplementation of NO, by the use of exogenous sources,restores ischemia-induced angiogenesis; and (iv) cardiovas-cular diseases associated with decreased NO synthesis dis-play an impairment in ischemia-induced angiogenesis.There are some crucial reports showing the link betweenShh and NO pathway. Indeed, Shh is involved in the regula-tion of NO release. NO-synthase (NOS) and VEGF aredownstream target of exogenous Shh signalling suggestingthat Shh can act as a modulator of the regulation of VEGFand NO production [138].In an experimental model of kidney ischemia reperfusion,Ozturk [118] suggests that NO reduces the renal dysfunctionFig. (2). Hh mechanism of action on angiogenesis. A, Direct mechanism: Hh directly stimulates endothelial cells to proliferate and to orga-nize the new vessels formation. B, Indirect mechanism: Hh acts on stroma cells which respond by producing proangiogenetic molecules suchas VEGF and Angiopoietin 1 and 2. Angiogenic molecules act on endothelial cells to induce the neovascularisation. The direct and indirectmodels are not exclusive; it is possible that Hh affects both the stromal and endothelial cells. tel-00441925,version1-17Dec2009 Sonic Hedgehog Pathway as a TargetCurrent Signal Transduction Therapy, 2009, Vol. 4, No. 1 7 associated to this pathology and they suggest that NO can actas a trigger to induce Shh and HIF-1 activities.On the course of liver ischemia and reperfusion in rats,inhibition of NO production upregulates Shh expression, inline with the hypothesis that Shh pathway is critical factor inthe pathophysiology of inflammation in the liver injury in-duced by ischemia/reperfusion [139].Recently, we have reported that MPs harbouring Shh areable to favour endothelial cell spreading and promote vascu-larisation in a model of mice hind limb ischemia by a mecha-nism sensitive to cyclopamine and trough the increase of NOproduction (unpublished data).Finally, oxidative stress induced by reactive species in-cluding superoxide, hydrogen peroxide, hydroxyl anion, andperoxynitrite, are biologically active oxygen derivatives thatare increasingly recognized to play major roles in vascularbiology through redox signalling [140-142]. ROS have beensuggested as important mediators of angiogenesis. ROSstimulate cell migration and proliferation in endothelial cells[143] and directly modulate VEGF expression and vascularsmooth muscle cell proliferation [144]. Of interest, ROS wasreported to stimulate post-ischemic revascularization at lowconcentrations but to inhibit it at high concentrations [145].A recent study of our laboratory shown that MPs bearingShh are able to restore endothelial dysfunction in a model ofmice coronary arteries subjected to ischemia/reperfusion.Shh carried by MPs restores endothelial injury probablythrough their dual ability to increase NO and reduce ROS.The concomitant effect of NO and ROS productions mightresult in an enhancement of the bioavailability of generatedNO by reducing oxidative stress and the subsequent scaveng-ing of NO. In addition, the increase in NO release is associ-ated with an enhancement of endothelial NO-synthase ex-pression and activity in human endothelial cells, as reflectedby both the increase in endothelial NO-synthase phosphory-lation, and changes in the expression and phosphorylation ofcaveolin-1. Altogether, these results indicate that Shh har-boured by MPs are able to modulate endothelial NO-synthase expression and activity and reduce oxidative stressin human endothelial cells, leading to a beneficial potentialeffect on the cardiovascular system [97].Hh pathway and cancerWhile Shh signalling is required for normal development,it has an equally important role in adulthood where muta-tions in the pathway give rise to a variety of tumours. Hhsignalling targets include genes that are important for cellproliferation proto-oncogenes as well as growth factors. De-regulation of the Hh pathway is a characteristic feature ofseveral pathologic states, including developmental syn-dromes with high predisposition to cancer [146, 147] (Table1). Indeed, Hh signalling has been implicated in the devel-opment of several human cancers, including medulloblas-toma, digestive tract tumours and basal cell carcinoma (Fig.3).Hh Signalling and MedulloblastomaMedulloblastomas (MB) are the most common malignantbrain tumours of childhood. A growing body of evidenceindicates that MB can arise by transformation of granuleneuron precursors. Shh pathway plays a critical role in cere-bellar development and its aberrant expression has beenidentified in MB. The target genes for Shh signalling afterreceptor activation include the transcription factors Gli-1, N-Myc and Ptc-1. These transcription factors can act as tumoursuppressors and negative regulators of the pathway. Pro-moter methylation of tumour suppressors plays a significantrole in different stage of tumour formation by gene silencing.For example in a mouse model of MB in which one allele ofPtc-1 is genetically disabled along with the p53, the remain-ing allele is naturally silenced by methylation [148]. Morerecently, it is found that the Ptc-1 promoter is methylated inovarian tumours, but not in basocellular carcinomas imply-ing differential Ptc-1 methylation as a contributing factor intumourigenesis, depending on tumour type [149]. Unexpect-edly, no methylation has been detected in the promoter imTable 1. Main Alterations in Shh Signalling in Several Types of Cancer Target GenesTumour TypeMechanism(s)References Nhlh1/NSCLMedulloblastomaNhlh1 activation by Gli-1[151]Gli-1Esophageal cancerDownregulation of Gli-1 expression[153]Ptc-1MedulloblastomaBasal-cell carcinomaInactivation of Ptc-1 by methylationMutation of Ptc-1[148][172]Gli-1, Gli-2, TGFGastric carcinomaActivation of Gli1 and Gli2 by TGF[157]ShhPancreatic carcinogenesisOverexpression of Shh[14, 161, 163]Gli-1, Ptc-1Colorectal cancerAberrant activation of Hh signaling (uncorrelatedexpression of Gli-1 with Ptc-1)[164] Hh, ERK1/2CholangiocarcinomaSimultaneous inhibition of Hh and ERK1/2 path-ways[175] Smo, c-mycHepatocarcinomaTumorigenesis by overexpression of Smo and c-myc (activated Hh pathway)[176]tel-00441925,version1-17Dec2009 8 Current Signal Transduction Therapy, 2009, Vol. 4, No. 1Porro et al. mediately upstream of Ptc-1B in either MB or control sam-ples. Future directions include examination of distal regionsof the Ptc-1B promoter as well as alternative exon variants,most notably the CpG island (genomic regions that contain ahigh frequency of CG dinucleotides), containing Ptc-1Cpromoter [150]. Identification of methylated genes that regu-late pathways common to many cancers may provide keyprognostic indicators and therapeutic targets.Furthermore, insulinoma-associated 1 and nescient helix–loop–helix 1 (Nhlh1)/NSCL1 are identified as new Hh targetgenes that are overexpressed in MB. Through the identifica-tion of functional Gli binding sites on the promoters ofmouse and human Nh1h1, Nhlh1 promoter is found to bebound and activated by Gli1 transcription factor. Remarka-bly, the expression of these genes is also upregulated inmouse and human Hh-dependent MBs, suggesting that theymay be either a part of the Hh-induced tumorigenic processor a specific feature of Hh-dependent tumour cells [151].Hh Signalling and Gastrointestinal CarcinogenesisaFunction of the Hh Signalling in Esophageal CancersThe Hh pathway plays a critical role in the developmentof the foregut. However, the role of the Hh pathway in pri-mary esophageal cancers is not well understood.Downregulation of Gli1 expression may be an importantmechanism by which KAAD-cyclopamine, found to be amore powerful derivative of cyclopamine [152], inhibitsgrowth and induces apoptosis in esophageal cancer cells.This finding suggests that targeted inhibition of the Hh sig-nalling by KAAD-cyclopamine or Shh neutralizing antibod-ies may be effective in chemoprevention and treatment ofesophageal cancers [153]. In addition, the Shh signallingpathway is extensively activated in esophageal cancer xeno-grafts and residual tumours after chemoradiotherapy and thetemporal kinetics of Hh signalling precedes increases in pro-liferation and tumour size during tumour regrowth. TheFig. (3). Hh pathway and cancer. The Hh pathway can be blocked at different levels, and Hh inhibitors could serves as anti-cancer agent.Inhibition of ligand activity has been reported with neutralising antibodies against Hh. A specific Smo inhibitor, cyclopamine. KAAD-cyclopamine, is identified as therapeutic agent to inhibit growth of esophageal cancer cells. Other compounds that block Gli activity could beused to treat wide of variety of Hh-dependent tumours. Ptc mutations are associated with basal-cell carcinoma (BCC), as well as with medul-loblastoma. Constitutively active forms of Smo are oncogenic and can function independently of ligand binding to Ptc, leading to BCC. Con-stitutively active forms of Smo are oncogenic and can function independently of ligand binding to Ptc, leading to BCC. An oncogenic formof Hh has been associated with BCC. Deregulation of Hh signalling has also been associated with pancreatic adenocarcinoma and oesophag-eal cancer. This highly stylized representation shows requirement for pathway activation in tumour formation and/or tumour cell growth, andthe functional interactions between the various components. In unstimulated cells, the activity of the transmembrane protein Smo is sup-pressed by the Hh receptor Ptc and the majority of the Gli transcription factors are present in their repressive (R) form. In stimulated cells,binding of Hh to Ptc activates Smo, which in turn shifts the Gli balance in favour of the activator (A) forms, resulting in the transcription ofHh target genes. Little is known about the molecular mechanisms by which Hh signalling is upregulated in these tumours.tel-00441925,version1-17Dec2009 Sonic Hedgehog Pathway as a TargetCurrent Signal Transduction Therapy, 2009, Vol. 4, No. 1 9 mechanism involved in the increase of proliferation of eso-phageal cancer cell lines is linked to an up-regulation of theG1-cyclin-Rb axis. Additionally, the same authors show thatblocking Hh signalling enhances radiation cytotoxicity ofesophageal cancer cells [154]. These results raise the ques-tion of what is a reliable marker of Hh pathway status incells where the pathway is not manipulated. Identification ofpost-transcriptional modifications, such as processing, sub-cellular localization or phosphorylation of some Hh pathwaymembers might predict more accurately the Hh pathway ac-tivity than expression levels. Clearly further studies areneeded to understand the possible involvement of this path-way in esophageal cancer before it can be considered as asuitable therapeutic target.bShh Signalling in Gastric CarcinomaRecent studies have suggested that constitutive activationof the Hh pathway in cancers of the digestive tract may con-tribute to the growth and maintenance of cancer.As mentioned previously, Hh proteins have long beenknown to act in the developing organism and to remain ac-tive in adult physiology (i.e., in the histostability of the gas-trointestinal tract) [155]. Previous studies have reported thataberrant activation of Shh signalling through Ptc-1 is fre-quently detected in cases of advanced gastric adenocarci-noma and that activation of this pathway is associated withpoorly differentiated gastric tumours [156].Furthermore, interactions between Shh and TGFpath-ways have been described in gastric cancer. Shh and TGF-family members are involved in numerous overlapping proc-esses during embryonic development, hair cycle, and cancer[158]. For TGF, this growth factor is a pleiotropic cytokinethat plays a critical role in the modulation of cell growth,differentiation, plasticity and migration. TGFreceptors,which function as tumour suppressors in normal and preneo-plastic tissues, acquire oncogenic functions during tumourprogression. In addition, TGFreceptors are mutated orexpressed at substantially attenuated levels in a variety ofhuman cancers and are correlated with the acquisition ofresistance to growth suppression by TGF[158]. In addi-tion, activation of MAP kinase and PI3K/Akt signalling cas-cades by TGFreceptors can also potentially contribute tocell migration and invasion. TGFsignalling is mediated byeither ALK1 or ALK5. Despite the critical role that Hh sig-nalling plays in the promotion of tumourigenesis, the mo-lecular and cellular mechanisms behind Hh regulation intumour metastatic behavior are unknown. Interestingly, Ber-tolino et al. [159] report that the Shh signalling pathway,acting through the TGF–ALK5 cascade, may selectivelycontribute to tumour cell motility and invasion in gastriccancer. In addition, they demonstrated that disruption ofTGF–ALK5 signalling by anti-TGFneutralizing anti-body or ALK5 kinase inhibitor results in suppression of Shh-mediated cell migration and invasion. These results indicatethat Shh promotes motility and invasiveness of gastric cancercells through TGF-mediated activation of the ALK5–Smad3 pathway. Additionally, these findings are the first to sug-gest a role for Shh signalling in the metastatic potential ofgastric cancer, thereby indicating potential therapeutic mo-lecular targets to decrease metastasis [157].Although activation of Hh signalling maintains prolifera-tion of some gastric cancer cells, Van Den Brink et al. [155]find that treatment of mice with cyclopamine increasedepithelial proliferation in vivo by 60–70%. Similarly, Fukayaet al. [160] show that treatment of primary cultures of nor-mal mouse gastric epithelial cells with cyclopamine increasetheir proliferation. This may indicate a difference in the roleof Hh signalling in cell cycle regulation between normal andcancer cells. More likely, one of the factors that may explainthe difference observed in the response of gastric carcinomacells and primary gastric epithelial cells in vivo and in cul-ture is probably linked to the use of pure population of cul-tured cancers cells rather than a combination of cells occur-ring in in vivo situation. [5].c-Hh Signalling in Initiation of Pancreatic CarcinogenesisShh is reported to play an essential role in the develop-ment of pancreatic cancer as well as pancreatic organogene-sis [148]. Thayer et al. [161] show that Shh is overexpressedin human preneoplastic pancreatic lesions and reported that amice model expressing Shh behind a pancreas specific pro-moter developed preneoplastic lesions. Shh has been de-tected in pancreatic cancers, and cyclopamine suppresses thegrowth of pancreatic cancer cells both in vitro and in vivo.Recently, it is reported that NFB contributes to Hhpathway activation through up-regulation of Shh expressionin pancreatic cancer cells [148]. In contrast, Hh signallingpathway and pancreatic cancer proliferation are suppressedwith Ptc-1 antibodies, indicating that Hh pathway may repre-sent a potential target in therapeutic against pancreatic can-cers [163].Recent evidence also indicates that deregulated signallingnot only causes tumour formation, but it is also required fortumour maintenance, as transformed cells continue to dependon Hh activity for survival and growth. Analysis of 26 hu-man pancreatic adenocarcinoma cell lines indicate that allcell lines express Hh target genes, and interestingly treatmentwith cyclopamine induces both apoptosis and loss of prolif-eration in 50% of the cell lines tested [14].dHh Signalling in Colorectal CancerIn contrast to other type of cancer, there is little evidencefor activation of Hh signalling at later stages of colorectalcarcinogenesis. Berman et al. [164], find that in contrast toproximal gastrointestinal cancer cells, colon cancer cells dosnot depend on Hh signalling for their survival. The authorsreport that the lack of sensitivity of colorectal cancer cells tocyclopamine correlates with the absence of active Hh signal-ling in these cells. This corroborates with the findings thatHh signalling is low or absent in undifferentiated colorectalcancer cells. Furthermore, Berman et al. [164] do not detectany Ptc-1 mRNA in colorectal cancer cells. However, Gli1expression correlates with Ptc-1 expression in all cell linesexamined except in the colorectal cancer cells. Correlationbetween Ptc-1 and Gli1 expression may be expected, as bothare transcriptional targets of Hh signalling. Also, since Smois expressed in colorectal cancer cells [165, 164] absence ofPtc-1 expression would be expected to activate the pathwayand would not result in low to absent activity of Hh signal-ling as observed by Berman et al. [164]. The loss of Hh sig-tel-00441925,version1-17Dec2009 10 Current Signal Transduction Therapy, 2009, Vol. 4, No. 1Porro et al. nalling in colorectal carcinogenesis is not yet completelyunderstood. It may be involved in the loss of epithelial dif-ferentiation that is a hallmark of adenomatous polyps. Chatelet al. [167] study seven colon cancer cell lines and find thatnone of the cell lines expressed all the key members of theHh pathway. They provide evidence that Ptc-1 and Gli1 tran-scripts levels are not significantly altered in cyclopamine-treated colon cancer cells, in contrast with the cyclopamine-responsive pancreatic cells. These results support the viewthat the aberrant activation of the Hh signalling pathway isnot common in colorectal cancer cell lines. In contrast, Qual-trough et al. [165] observe increased cell death in a varietyof colonic epithelial cell lines treated with cyclopamine. Cur-rently, there is no explanation for this difference, but thesensitivity of the viability of colorectal cancer cells to theeffects of cyclopamine may depend on cell culture condi-tions. Further studies are needed to sort out these differences.Shh in Basal-Cell CarcinomaBasal-cell carcinomas (BCC), account for 90% of all skincancers, represent the commonest human cancer in fair skin-ned populations, and they are clearly associated with consti-tutive activation of Shh signalling.Mutations in the Shh receptor, Ptc-1, are found in famil-ial [168, 169] and sporadic [170, 171] forms of BCC. In thedeveloping epidermis, Shh signalling is required to maintainthe balance between cell proliferation and differentiation.Ptc-1 plays an important tumour suppressor function in themammalian epidermis. This conclusion is based on the factthat spontaneous allele of Ptc-1, Ptc-1mes, which encodes amutant Ptc-1 protein lacking the last 220 amino acid residue,has a critical role in epidermal hyperplasia [172]. Indeed,Nieuwenhuis et al. [173] have bypassed the early embryoniclethality associated with the Ptc-1null mutation and demon-strate an important function of Ptc-1 in adult skin. The ob-servation that Ptc-1mes/mes mice possess excess skin sug-gests a potential role for the CTD (carboxy-terminal domain)of Ptc-1 in skin development. A detailed analysis of epider-mal development and homeostasis in Ptc-1mes/mes mice hasshown that embryonic skin development appears normal.Interestingly, Ptc-1mes/mes mice display epidermal hyper-plasia beginning at postnatal day 12. Importantly, an expan-sion of the epidermal stem cell compartment in Ptc-1mes/mes mice is described. These results suggest that theCTD of Ptc-1 is not required for epidermal development inembryonic skin, but is necessary for epidermal homeostasisin adult skin [173].Hh in Cholangiocarcinoma (CCA) and Hepatocarcinoge-nesisHh pathway deregulation has been reported in CCA andhepatoblastoma cell lines [174]. Jinawath et al. [175] suggestthat the Hh and ERK1/2 pathways are important for CCAcell proliferation, and simultaneous inhibition of the twopathways may lead to stronger decreases in cell growth andviability in a subset of CCA cases. Sicklick et al. (2006)[176] show that in hepatocarcinoma, overexpression of theSmo protooncogene, as well as an increase in the stoichio-metric ratio of Smo to Ptc-1 mRNA levels, correlate withtumour size, can be used as a prognostic indicator in hepato-carcinoma. They also demonstrated that hepatocarcinomacell lines (HepG2 and Hep3B) express Hh pathway compo-nents and activate Hh transcriptional targets. Hh pathwayactivation may occur as an early event during the evolutionof hepatic neoplasia. These data support the hypothesis thatHh signalling is deregulated in human hepatocarcinogenesis.The authors report that overexpression and/or tumorigenicactivation of the Smo protooncogene mediates c-myc over-expression which plays a critical role in hepatocarcinogene-sis. The results also suggest that Smo is a prognostic factorin hepatocarcinoma tumourigenesis [176].FUTURE DIRECTIONSData in the literature suggest that an increase of neovas-cularisation through the Shh pathway activation might havebeneficial effects on pathologies associated with ischemiccomplications whereas in cancers, novel potential therapeuticstrategies addresses against Shh pathway may lead to re-duced tumour development.Concerning neovascularisation, it has been recently re-ported that inhibition of endogenous Shh pathway may ame-liorate cardiac function after myocardial ischemia. This ef-fect is associated with reduced apoptosis and fibrosis andincreased vascularisation.Thus, a dual ability of Shh pathway in cardiac ischemiahas been proposed by these authors [177]. On one hand, highexogenous level of Hh may favour tissue repair. On the otherhand, endogenous Hh may act as a deleterious factor. Thesedata may explain, at least in part, why exogenous therapywith recombinant Hh proteins or genetic therapy with ade-novirus are effective in inducing neovascularisation of tissuerepair. Furthermore, other potential therapies for exampleusing MPs harbouring Shh seem promising, as shown inischemic animal models and in vitro. However, clinical stud-ies need to be performed in order to validate these ap-proaches.Also, the use and development of specific agonists mayconstitute another potential tool to activate Hh pathway. Inthis respect, effort of the pharmaceutical companies will benecessary in order to evolve towards a large spectrum of newdrugs targeting, specifically endothelial cells and endothelialprogenitors in order to favour their ability to promote angio-genesis.Taking in consideration that in several type of tumoursHh signalling is upregulated, the use of cyclopamine or itsderivates may be an interesting tool for decrease tumourformation. Parker and Inghram (2008) [178] have reviewedthese aspects and they consider that the pathway downstreamof Smo, such as the Gli transcription factors may be the tar-gets of new drugs developed in order to limit cancer growth.In addition, it should be noted that the concomitant inhibitionof pathways that can be implicated separately in several can-cer development may increase the success of tumour therapy.For instance, a correlation between estrogen receptor alpha(ER ) and Shh expression is found in the breast cancer tis-sues. Moreover estrogen triggers Shh up-regulation and inhi-bition of ER suppresses this effect, suggesting that ERregulates the Hh pathway though Shh induction [179]. Con-tel-00441925,version1-17Dec2009 Sonic Hedgehog Pathway as a TargetCurrent Signal Transduction Therapy, 2009, Vol. 4, No. 1 11 comitant inhibition of both pathways (ER and Shh) maylead to a reduction of breast cancer development.Taking together, one can advanced the prediction that ourunderstanding of Hh pathway mechanisms and the participa-tion of Hh pathway in angiogenesis related disease is a grow-ing area of research. Together with progress in technologiessuch as cell and gene therapy and diagnostic, this will aid inapplying our knowledge to clinical practice and will offer usa means to fight against ischemic and cancer diseases in theadult.CONCLUSIONThe Hh family of proteins is powerful morphogen medi-ating embryonic development as well as adult morphogene-sis. The majority studies have focused on the role of Hh fam-ily members during embryogenesis, they have shown themorphogen implicated in the regulation of epithelial-mesen-chymal interaction crucial to limb, lung, gut, hair follicle,and bone formation, including a possible role during vascu-larisation of certain embryonic tissues [115].In addition to its role in patterning the developing em-bryo, several recent studies indicate that Hh signalling isinvolved in neovascularisation in part by upregulation ofmultiple families of angiogenic growth factors [83]. Theinnate Hh pathway is activated after myocardial ischemiaand Shh gene therapy may have therapeutic potential underpathological conditions that need angiogenesis such as lowerextremity ischemia, myocardial ischemia, and in cerebralischemia.Even if Shh therapy may have positive effects in angio-genesis-related diseases, there is a significant concern thatthe induction of angiogenesis may increase the risk of neo-plastic diseases. Indeed, there are also data supporting thistheory inasmuch upregulation of Shh signalling has beenreported in human and animal models of basal cell carci-noma. This might suggest that transfection of Shh could belimited as a clinical strategy for ischemia, because of a po-tential risk for cancer induction.However, it should be noted that reports regarding thecarcinogenic effect of Shh have been conducted in transgenicor mutated mice and the observation that a mutation in Shhpathway is found in a percentage of patient with basal carci-noma. It is probable that the high dose and the long-termexposure of Shh resulting from the genetic models, as well asthe deregulated signalling that occurs in the presence of thehuman mutation, are factors in the development of cancer.Thus, it is possible that the lower dose and the short-termexposure to unmutated Shh, would not generate a similardegree of risk, if any [119].However Hh signalling is not upregulated in all forms ofcancer. 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Received: July 22, 2008 Revised: August 19, 2008 Accepted: September 04, 2008 tel-00441925,version1-17Dec2009