Lignin biosynthesis in Norway spruce: from a model system to the tree

Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the lignin-forming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the up-regulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses.

Management of semi-natural grasslands for butterfly and moth communities

This work focuses on the factors affecting species richness, abundance and species composition of butterflies and moths in Finnish semi-natural grasslands, with a special interest in the effects of grazing management. In addition, an aim was set at evaluating the effectiveness of the support for livestock grazing in semi-natural grasslands, which is included in the Finnish agri-environment scheme. In the first field study, butterfly and moth communities in resumed semi-natural pastures were com-pared to old, annually grazed and abandoned previous pastures. Butterfly and moth species compo-sition in restored pastures resembled the compositions observed in old pastures after circa five years of resumed cattle grazing, but diversity of butterflies and moths in resumed pastures remained at a lower level compared with old pastures. None of the butterfly and moth species typical of old pas-tures had become more abundant in restored pastures compared with abandoned pastures. There-fore, it appears that restoration of butterfly and moth communities inhabiting semi-natural grass-lands requires a longer time that was available for monitoring in this study. In the second study, it was shown that local habitat quality has the largest impact on the occurrence and abundance of butterflies and moths compared to the effects of grassland patch area and connec-tivity of the regional grassland network. This emphasizes the importance of current and historical management of semi-natural grasslands on butterfly and moth communities. A positive effect of habitat connectivity was observed on total abundance of the declining butterflies and moths, sug-gesting that these species have strongest populations in well-connected habitat networks. Highest species richness and peak abundance of most individual species of butterflies and moths were generally observed in taller grassland vegetation compared with vascular plants, suggesting a preference towards less intensive management in insects. These differences between plants and their insect herbivores may be understood in the light of both (1) the higher structural diversity of tall vegetation and (2) weaker tolerance of disturbances by herbivorous insects due to their higher trophic level compared to plants. The ecological requirements of all species and species groups inhabiting semi-natural grasslands are probably never met at single restricted sites. Therefore, regional implementation of management to create differently managed areas is imperative for the conservation of different species and species groups dependent on semi-natural grasslands. With limited resources it might be reasonable to focus much of the management efforts in the densest networks of suitable habitat to minimise the risk of extinction of the declining species.

Suomenlinnan Kustaanmiekan ketokasvillisuus ja siihen vaikuttavat tekijät

Suomenlinna on yksi Helsingin suosituimmista matkailu- ja kulttuurinähtävyyksistä. Kustaanmiekan, samoin kuin koko Suomenlinnan luonto on muodostunut perinteisestä suomalaisesta saaristoluonnosta ja vuosisatojen saatossa paikalle tulleista linnoituksien kasvistosta. Saaren vaihtelevien elinympäristöjen johdosta alueen kasvillisuus on hyvin rikasta. Linnoituksien monet kasvilajit ovat tulleet tulokaskasveina eri puolilta Eurooppaa sekä Venäjältä. Suurin osa Suomenlinnan alueesta on kallioketoa ja tämän lisäksi myös valliketoa, joista molemmat kuuluvat suojeltaviin alueisiin. Kustaanmiekan niityillä kasvaa keto- ja paahdelajeja, kuten harvinaista ketonoidanlukkoa (Botrychium lunaria L.) sekä ketoneilikkaa (Dianthus deltoides L.). Tämän tutkimuksen ensisijaisena tarkoituksena oli kartoittaa Kustaanmiekan alueen kesäkauden 2009 ketokasvilajisto ja eri putkilokasvilajien runsaus. Tutkimuksessa selvitettiin myös maaperätekijöiden ja alueen hoitohistorian mahdollista vaikutusta ketokasvilajistoon. Tutkimuksessa kartoitettiin kymmenen eri kedon kasvillisuus Suomenlinnan Kustaanmiekan linnoitusalueella. Kedot sijaitsivat eri puolilla Kustaanmiekkaa, sellaisilla paikoilla, missä ketokasvillisuus oli runsainta. Maastotyöt suoritettiin kesä- ja heinäkuussa laskemalla jokaisen kedon ruutujen putkilokasvien peittävyydet sekä listaamalla ylös myös ruutujen ulkopuoliset kevät- ja loppukesän kukkijat touko- ja elokuussa. Maaperän ominaisuuksien määrittämiseksi otettiin kultakin kedolta pintamaanäytteet elokuussa. Muita tutkittuja muuttujia olivat maapinnan kaltevuus sekä sammalen, karikkeen, paljaan maan, kenttäkasvillisuuden pohjakerros ja kallion osuus tutkimusruuduilla. Ketojen kasvillisuuden keskimääräinen korkeus mitattiin kesä- ja heinäkuussa. Kasvistossa oli selviä eroavaisuuksia ketojen välillä. Kasvilajien määrä vaihteli ketojen kokonaislajimäärän ollessa 40-60 kasvilajia. Yhteensä kedoilta löytyi 120 eri putkilokasvilajia, joista useimmat kukkivat sekä kesä- että heinäkuussa. Ketojen kasvilajimäärä vaihteli yhdellä neliömetrillä 6,3-13,6 kasvilajiin, minkä lisäksi Shannon-Wienerin diversiteetti-indeksi vaihteli 1,4-2,3 arvon välillä. Yleisimpiä lajeja, joita kedoilla tavattiin, olivat muun muassa siankärsämö (Achillea millefolium L.), koiranheinä (Dactylis glomerata L.), juolavehnä (Elymus repens L.) ja hopeahanhikki (Potentilla argentea L.). Alueella kasvoi myös muutamia sotatulokaslajeja kuten harmiota (Berteroa incana L.), ukonpalkoa (Bunias orientalis L.) ja karvahorsmaa (Epilobium hirsutum L.). Maaperätekijöillä, kuten suurella fosforin pitoisuudella ei ollut vaikutusta kasvilajien määrään kedoilla. Vain maan pH ja johtoluku korreloivat positiivisesti ketojen kasvillisuuden korkeuden kanssa. Vaikka tulosten perusteella ketojen hoidolla ei ollut vaikutusta ketojen kasvillisuuden määrään, voidaan kuitenkin olettaa oikeanlaisen hoidon parantavan tyypillisten ketokasvien kilpailukykyä muita niittykasveja kohtaan.

Luonnonhoitopellot maatalousympäristön luonnon monimuotoisuuden edistämisessä

Luonnonhoitopellot lisättiin uutena vapaaehtoisena toimenpiteenä maatalouden ympäristötukeen vuonna 2009. Luonnonhoitopeltoihin kuuluvat monivuotiset nurmipellot sekä niitty-, riista- ja maisemakasveilla kylvetyt pellot. Toimenpiteen avulla pyritään suojelemaan ja lisäämään maatalousympäristön luonnon monimuotoisuutta sekä muun muassa vähentämään maatalouden ravinnehuuhtoumia. Vuonna 2010 luonnonhoitopeltoja oli yli seitsemän prosenttia Suomen viljelyalasta. Luonnonhoitopeltojen ympäristövaikutusten arvioiminen ja toimenpiteen kehittäminen on tärkeää, jotta toimenpiteeseen käytettävät varat eivät valu hukkaan. Tämän tutkimuksen tavoitteena oli selvittää, millainen merkitys luonnonhoitopelloilla on maatalousympäristön monimuotoisuuden kannalta; millaiset luonnonhoitopellot ovat monimuotoisuuden kannalta arvokkaimpia; ja kuinka toimenpidettä kannattaa kehittää. Kysymyksiin pyrittiin vastaamaan tutkimalla putkilokasvilajistoa, -lajirikkautta ja kasvillisuuden rakennetta erilaisilla luonnonhoitopelloilla, sekä vertaamalla luonnonhoitopeltojen kasvillisuutta pientareiden ja niittyjen kasvillisuuteen. Maastotyö luonnonhoitopelloilla tehtiin kesällä 2010 Uudenmaan ja Pohjois-Pohjanmaan ELY-keskusalueilla. Lisäksi tutkimuslohkoista kerättiin tietoja viljelijäkyselyn kautta. Niittypellot osoittautuivat tutkimuksessa lajirikkaimmaksi luonnonhoitopeltotyypiksi, mikä selittynee lähinnä sillä, että ne kylvetään heikosti kilpailukykyisellä siemenseoksella. Nurmi- ja niittypelloilla lohkon viljavuus korreloi negatiivisesti lajirikkauden kanssa. Lajikoostumukseltaan kaikki luonnonhoitopellot eroavat toisistaan sekä maatalousympäristön puoliluonnontilaisista elinympäristöistä, ja lisäävät siten monimuotoisuutta maisematasolla. Luonnonhoitopeltojen lajisto ei ole kuitenkaan suojelun kannalta erityisen arvokasta. Toimenpiteen maatalousympäristöä rikastuttavaa vaikutusta vähentää, että tällä hetkellä valtaosa luonnonhoitopelloista on monivuotisia nurmipeltoja. Luonnonhoitopeltojen siemenseoksia kehittämällä, perustamis- ja hoitomenetelmiä tutkimalla ja neuvontaan panostamalla voidaan lisätä luonnonhoitopeltojen arvoa niin luonnon, viljelijän kuin yhteiskunnankin kannalta.

Suokasvillisuuden vaste muuttuvaan ilmastoon: kokeellinen tutkimus kuivumisen vaikutuksesta

Soilla on merkittävä rooli ilmastonmuutoksen hillitsemisessä suuren hiilivarastonsa sekä ekosysteemin ja ilmakehän välisen kaasunvaihdon ansiosta. Ilmastonmuutoksen ennustetaan vaikuttavan suokasvillisuuteen ja suon toimintaan epäsuorasti. Vedenpinnan ennustetaan laskevan 14–21 cm johtuen kasveista ja avoimilta pinnoilta tapahtuvan haihdunnan lisääntymisestä lämpötilan noustessa, mikäli sadanta ei lisäänny. Aiemmat vedenpinnan laskun jälkeistä kasvillisuutta seuranneet tutkimukset ovat osoittaneet, että putkilokasvit hyötyvät alhaisemmasta vedenpinnan tasosta ja että kuljuun sopeutuneet rahkasammalet kärsivät kuivuneista oloista. Kasvillisuuden runsaussuhteiden muuttumisen lisäksi kasviyhteisöjen monimuotoisuus vähenee. Erityisen herkkiä vedenpinnan laskulle ovat olleet välipinta- ja kuljurahkasammalet ja sarat. Funktionaalisten kasviryhmien vasteiden selvittämiksesi käytettiin BACI (before-after-control-impact) –tutkimusotetta. Tutkimuksessa oli kolme verrokkialaa ja kolme käsittelyalaa, joissa vedenpintaa oli laskettu 14–21 senttimetriin. Lisäksi vertailukohdaksi tutkimuksessa oli mukana kolme alaa, joissa oli tehty metsäojitus n. 50 vuotta sitten. Nämä toistot sijaitsivat meso-, oligo ombrotrofisilla suotyypeillä Oriveden Lakkasuolla. Kasvillisuus kartoitettiin ja vedenpinnat mitattiin aloilta ennen käsittelyä vuonna 2000 sekä vuosina 2001–2003 ja 2009. Aineisto analysoitiin TWINSPAN- (PC-Ord), PRC ja DCA (CANOCO)-monimuuttujamenetelmillä. Tulokset osoittivat, että verrokki- ja käsittelyalat olivat samanlaisia lähtökohdiltaan, joten niitä voitiin käsittelyn jälkeen verrata toisiinsa. Kasvillisuuden rakenne vaihteli vuosien välillä myös verrokkialoilla, mikä osoittaa kasvien sopeutumiskyvyn muuttuviin sääoloihin (lämpötila, sademäärä). Vuosi 2003 erottui tutkimuksessa alhaisella vedenpinnantasolla, mutta toisaalta myös ainavihantien varpujen suuren peittävyyden osalta. Vuoteen 2009 mennessä kasvillisuuden erityisesti sarojen peittävyys väheni. Ravinteikkaimmilla toistoilla kasvillisuuden vasteet vaikuttivat olevan vahvemmat kuin vähäravinteisilla toistoilla. Kasviryhmistä kulju- ja välipintasammalilla oli vahvimmat vasteetvedenpinnan laskuun ja mätäslajeilla heikoimmat. Tulosten mukaan kasviryhmien vasteet vaihtelevat riippuen tarkasteltavasta aikajaksosta: ensimmäiset kolme vuotta käsittelyn jälkeen suo oli häiriötilassa ja vasta sen jälkeen kasvillisuus sopeutui muuttuneisiin oloihin.

Lebanese Plants and Plant-Derived Compounds Against Colon Cancer

Colorectal cancer (CRC) is a major health concern and demands long-term efforts in developing strategies for screening and prevention. CRC has become a preventable disease as a consequence of a better understanding of colorectal carcinogenesis. However, current therapy is unsatisfactory and necessitates the exploration of other approaches for the prevention and treatment of cancer. Plant based products have been recognized as preventive with regard to the development of colon cancer. Therefore, the potential chemopreventive use and mechanism of action of Lebanese natural product were evaluated. Towards this aim the antitumor activity of Onopordum cynarocephalum and Centaurea ainetensis has been studied using in vitro and in vivo models. In vitro, both crude extracts were non cytotoxic to normal intestinal cells and inhibited the proliferation of colon cancer cells in a dose-dependent manner. In vivo, both crude extracts reduced the number of tumors by an average of 65% at weeks 20 (adenomas stage) and 30 (adenocarcinomas stage). The activity of the C. ainetensis extract was attributed to Salograviolide A, a guaianolide-type sesquiterpene lactone, which was isolated and identified through bio-guided fractionation. The mechanism of action of thymoquinone (TQ), the active component of Nigella sativa, was established in colon cancer cells using in vitro models. By the use of N-acetyl cysteine, a radical scavenger, the direct involvement of reactive oxygen species in TQ-induced apoptotic cells was established. The analytical detection of TQ from spiked serum and its protein binding were evaluated. The average recovery of TQ from spiked serum subjected to several extraction procedures was 2.5% proving the inability of conventional methods to analyze TQ from serum. This has been explained by the extensive binding (>98%) of TQ to serum and major serum components such as bovine serum albumin (BSA) and alpha-1-acid glycoprotein (AGP). Using mass spectrometry analysis, TQ was confirmed to bind covalently to the free cysteine in position 34 and 147 of the amino acid sequence of BSA and AGP, respectively. The results of this work put at the disposal for future development new plants with anti-cancer activities and enhance the understanding of the pharmaceutical properties of TQ, a prerequisite for its future clinical development.

Determinants of vascular cognitive impairment : White matter lesions, brain atrophy and their neuropsychological correlates

The concept of vascular cognitive impairment (VCI) covers a wide spectrum of cognitive dysfunctions related to cerebrovascular disease. Among the pathophysiological determinants of VCI are cerebral stroke, white matter lesions and brain atrophy, which are known to be important risk factors for dementia. However, the specific mechanisms behind the brain abnormalities and cognitive decline are still poorly understood. The present study investigated the neuropsychological correlates of particular magnetic resonance imaging (MRI) findings, namely, medial temporal lobe atrophy (MTA), white matter hyperintensities (WMH), general cortical atrophy and corpus callosum (CC) atrophy in subjects with cerebrovascular disease. Furthermore, the cognitive profile of subcortical ischaemic vascular disease (SIVD) was examined. This study was conducted as part of two large multidisciplinary study projects, the Helsinki Stroke Aging Memory (SAM) Study and the multinational Leukoaraiosis and Disability (LADIS) Study. The SAM cohort consisted of 486 patients, between 55 and 85 years old, with ischaemic stroke from the Helsinki University Hospital, Helsinki, Finland. The LADIS Study included a mixed sample of subjects (n=639) with age-related WMH, between 65 and 84 years old, gathered from 11 centres around Europe. Both studies included comprehensive clinical and neuropsychological assessments and detailed brain MRI. The relationships between the MRI findings and the neuropsychological test performance were analysed by controlling for relevant confounding factors such as age, education and other coexisting brain lesions. The results revealed that in elderly patients with ischaemic stroke, moderate to severe MTA was specifically related to impairment of memory and visuospatial functions, but mild MTA had no clinical relevance. Instead, WMH were primarily associated with executive deficits and mental slowing. These deficits mediated the relationship between WMH and other, secondary cognitive deficits. Cognitive decline was best predicted by the overall degree of WMH, whereas the independent contribution of regional WMH measures was low. Executive deficits were the most prominent cognitive characteristic in SIVD. Compared to other stroke patients, the patients with SIVD also presented more severe memory deficits, which were related to MTA. The cognitive decline in SIVD occurred independently of depressive symptoms and, relative to healthy control subjects, it was substantial in severity. In stroke patients, general cortical atrophy also turned out to be a strong predictor of cognitive decline in a wide range of cognitive domains. Moreover, in elderly subjects with WMH, overall CC atrophy was related to reduction in mental speed, while anterior CC atrophy was independently associated with frontal lobe-mediated executive functions and attention. The present study provides cross-sectional evidence for the involvement of WMH, MTA, general cortical atrophy and CC atrophy in VCI. The results suggest that there are multifaceted pathophysiological mechanisms behind VCI in the elderly, including both vascular ischaemic lesions and neurodegenerative changes. The different pathological changes are highly interrelated processes and together they may produce cumulative effects on cognitive decline.

Milk casein-derived tripeptides : A special focus on blood pressure and vascular function

Increased consumption of low-fat milk products is inversely associated with the risk of hypertension. The beneficial effect of milk on blood pressure is attributed to high calcium and potassium content but also to specific peptide sequences, which are cleaved from milk protein during gastrointestinal digestion, fermentation of milk with proteolytic starter cultures or enzymatic hydrolysis. Milk products fermented with Lactobacillus helveticus contain casein-derived tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro), which have been shown to possess antihypertensive effects in humans and in experimental animals. The aim of the present series of studies was to investigate the effects of tripeptides Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them on vascular function and blood pressure and to elucidate the mechanisms behind them by using different experimental models of hypertension. Another aim was to characterize the acute effects of tripeptides on blood pressure and arterial stiffness in mildly hypertensive humans. Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them attenuated the development of hypertension in two experimental models of hypertension, spontaneously hypertensive rat (SHR) and type 2 diabetic Goto-Kakizaki (GK) rat fed with high-salt diet. Significant differences in systolic blood pressure (SBP) were seen after 8 weeks treatment with tripeptide-containing products compared to control product. Plant sterols did not enhance this effect. Two differently produced tripeptide powders produced a similar attenuating effect on SBP in SHR. In mildly hypertensive subjects, a single administration of tripeptide- and plant sterol-containing fermented milk product decreased both SBP and diastolic blood pressure (DBP) over a period of 8 hours. Protective effect of tripeptides Ile-Pro-Pro and Val-Pro-Pro and fermented milk products containing them on vascular function was demonstrated in in vitro studies and long-term experimental studies. The effect was shown to be endothelium-dependent and possibly involving endothelium-derived hyperpolarizing factor (EDHF). In the clinical study, single administration of tripeptide-containing fermented milk product did not affect measures of arterial stiffness. Long-term treatment with fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro inhibited angiotensin-converting enzyme (ACE) and decreased aldosterone levels thus showing beneficial effects on the renin-angiotensin system (RAS) in SHR and GK. No changes in the components of RAS were observed by the single administration of the same product in mildly hypertensive subjects. Increased levels of cGMP, NOx and citrulline suggest increased nitric oxide (NO) production by the tripeptides. Taken together, Ile-Pro-Pro and Val-Pro-Pro -containing products attenuate the development of hypertension after long-term treatment in experimental models of hypertension and possess an acute antihypertensive effect in mildly hypertensive subjects. In addition, these tripeptides show endothelium-mediated beneficial effects on vascular function. Attenuation of blood pressure increase by the tripeptides in experimental animals involves RAS, but its role in the antihypertensive effect in humans remains to be elucidated.

VEGFR-3 and Tie pathways in vascular network formation

The blood and lymphatic vascular systems are essential for life, but they may become harnessed for sinister purposes in pathological conditions. For example, tumors learn to grow a network of blood vessels (angiogenesis), securing a source of oxygen and nutrients for sustained growth. On the other hand, damage to the lymph nodes and the collecting lymphatic vessels may lead to lymphedema, a debilitating condition characterized by peripheral edema and susceptibility to infections. Promoting the growth of new lymphatic vessels (lymphangiogenesis) is an attractive approach to treat lymphedema patients. Angiopoietin-1 (Ang1), a ligand for the endothelial receptor tyrosine kinases Tie1 and Tie2. The Ang1/Tie2 pathway has previously been implicated in promoting endothelial stability and integrity of EC monolayers. The studies presented here elucidate a novel function for Ang1 as a lymphangiogenic factor. Ang1 is known to decrease the permeability of blood vessels, and could thus act as a more global antagonist of plasma leakage and tissue edema by promoting growth of lymphatic vessels and thereby facilitating removal of excess fluid and other plasma components from the interstitium. These findings reinforce the idea that Ang1 may have therapeutic value in conditions of tissue edema. VEGFR-3 is present on all endothelia during development, but in the adult its expression becomes restricted to the lymphatic endothelium. VEGF-C and VEGF-D are ligands for VEGFR-3, and potently promote lymphangiogenesis in adult tissues, with direct and remarkably specific effects on the lymphatic endothelium in adult tissues. The data presented here show that VEGF-C and VEGF-D therapy can restore collecting lymphatic vessels in a novel orthotopic model of breast cancer-related lymphedema. Furthermore, the study introduces a novel approach to improve VEGF-C/VEGF-D therapy by using engineered heparin-binding forms of VEGF-C, which induced the rapid formation of organized lymphatic vessels. Importantly, VEGF-C therapy also greatly improved the survival and integration of lymph node transplants. The combination of lymph node transplantation and VEGF-C therapy provides a basis for future therapy of lymphedema. In adults, VEGFR-3 expression is restricted to the lymphatic endothelium and the fenestrated endothelia of certain endocrine organs. These results show that VEGFR-3 is induced at the onset of angiogenesis in the tip cells that lead the formation of new vessel sprouts, providing a tumor-specific vascular target. VEGFR-3 acts downstream of VEGF/VEGFR-2 signals, but, once induced, can sustain angiogenesis when VEGFR-2 signaling is inhibited. The data presented here implicate VEGFR-3 as a novel regulator of sprouting angiogenesis along with its role in regulating lymphatic vessel growth. Targeting VEGFR-3 may provide added efficacy to currently available anti-angiogenic therapeutics, which typically target the VEGF/VEGFR-2 pathway.