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.Verisuonet ja imusuonet ovat elämälle välttämättömiä järjestelmiä. Verisuonet kuljettavat kudoksiin happea ja ravinteita, sekä poistavat niistä haitallisia aineenvaihduntatuotteita. Verisuonten uudismuodostus (angiogeneesi) on myös välttämätöntä syöpäkasvaimen kehittymiselle, ja angiogeneesin estäminen onkin houkutteleva tapa estää syövän kasvua. Imusuonet puolestaan keräävät kudoksista nestettä, valkuaisaineita ja puolustusjärjestelmän soluja. Imusuonten toiminnan estyminen johtaa krooniseen turvotustautiin, lymfedeemaan, johon ei tällä hetkellä ole olemassa parantavaa hoitoa. Tämän väitöskirjatutkimuksen tavoitteena oli kehittää uusia hoitomuotoja lymfedeemaan, sekä selvittää menetelmiä estää syöpäkasvaimen verisuonten kasvua tautimalleissa. Tutkimuksen ensimmäinen osatyö osoitti, että angiopoietiini-1-kasvutekijä aiheuttaa imusuonten uudismuodostusta (lymfangiogeneesiä). Esimerkiksi rintasyöpäpotilailta joudutaan usein poistamaan kainalon alueen imusolmukkeet, mikä johtaa imunestekierron häiriöön ja usein yläraajan lymfedeemaan. Tutkimuksen toinen osatyö näytti, että kainaloalueelle voidaan kasvattaa uudet toimivat imusuonet VEGF-C-kasvutekijän avulla. Lisäksi VEGF-C-hoidon avulla voitiin siirtää vaurioalueelle uusia imusolmukkeita, ja kytkeä nämä paikalliseen imusuonijärjestelmään. Kolmannessa osatyössä osoitettiin, että muokkaamalla VEGF-C:n ominaisuuksia sitoutua soluväliaineeseen imusuonten järjestäytymistä toimintakuntoisiksi voitiin nopeuttaa. Nämä tutkimukset muodostavat pohjan kasvutekijähoidon kehittämiseksi lymfedeemapotilaille. Neljännessä osatyössä näytettiin VEGF-C:n reseptorin VEGFR-3:n säätelevän verisuonten uudismuodostusta niin yksilönkehityksen kuin syöpäkasvaimen kasvunkin aikana. VEGFR-3:n estäminen vähensi verisuonten määrää kokeellisissa kasvaimissa, sekä hidasti kasvainten kasvua. Huomattavaa oli, että VEGFR-3:n estäjä lisäsi entisestään tunnetun angiogeneesin estäjän tehoa. Lisäksi havaittiin, että VEGFR-3:n estäminen ei johtanut merkittäviin haittavaikutuksiin. Tutkimuksessa selvitettiin lisäksi VEGFR-3:n säätelymekanismeja. Näiden tulosten perusteella VEGFR-3 on lupaava kohdemolekyyli angiogeneesiä estävien lääkeaineiden kehitykselle

Development and validation of a high-content screening assay for inhibitors of enteropathogenic E. coli adhesion

Enteropathogenic E. coli (EPEC) causes intestinal infections leading to severe diarrhea. EPEC attaches to the host cell causing lesions to the intestinal epithelium coupled with the effacement of microvilli. In the process, actin accumulates into a pedestal-like structure under bacterial microcolonies. We designed an automated fluorescence microscopy-based screening method for discovering compounds capable of inhibiting EPEC adhesion and virulence using aurodox, a type three secretion system (T3SS) inhibitor, as a positive control. The screening assay employs an EPEC strain (2348/69) expressing a fluorescent protein and actin staining for monitoring the bacteria and their pedestals respectively, analyzing these with a custom image analysis pipeline. The assay allows for the discovery of compounds capable of preventing the formation of pathogenic actin rearrangements. These compounds may be interfering with virulence-related molecular pathways relevant for developing antivirulence leads.Peer reviewe

Expected impact of MRI-related interreader variability on ProScreen prostate cancer screening trial: a pre-trial validation study

Background: The aim of this study is to investigate the potential impact of prostate magnetic resonance imaging (MRI) -related interreader variability on a population-based randomized prostate cancer screening trial (ProScreen). Methods: From January 2014 to January 2018, 100 men aged 50-63 years with clinical suspicion of prostate cancer (PCa) in Helsinki University Hospital underwent MRI. Nine radiologists individually reviewed the pseudonymized MRI scans of all 100 men in two ProScreen trial centers. All 100 men were biopsied according to a histological composite variable comprising radical prostatectomy histology (N = 38) or biopsy result within 1 year from the imaging (N = 62). Fleiss' kappa (kappa) was used to estimate the combined agreement between all individual radiologists. Sample data were subsequently extrapolated to 1000-men subgroups of the ProScreen cohort. Results: Altogether 89% men of the 100-men sample were diagnosed with PCa within a median of 2.4 years of follow-up. Clinically significant PCa (csPCa) was identified in 76% men. For all PCa, mean sensitivity was 79% (SD +/- 10%, range 62-96%), and mean specificity 60% (SD +/- 22%, range 27-82%). For csPCa (Gleason Grade 2-5) MRI was equally sensitive (mean 82%, SD +/- 9%, range 67-97%) but less specific (mean 47%, SD +/- 20%, range 21-75%). Interreader agreement for any lesion was fair (kappa 0.40) and for PI-RADS 4-5 lesions it was moderate (kappa 0.60). Upon extrapolating these data, the average sensitivity and specificity to a screening positive subgroup of 1000 men from ProScreen with a 30% prevalence of csPCa, 639 would be biopsied. Of these, 244 men would be true positive, and 395 false positive. Moreover, 361 men would not be referred to biopsy and among these, 56 csPCas would be missed. The variation among the radiologists was broad as the least sensitive radiologist would have twice as many men biopsied and almost three times more men would undergo unnecessary biopsies. Although the most sensitive radiologist would miss only 2.6% of csPCa (false negatives), the least sensitive radiologist would miss every third. Conclusions: Interreader agreement was fair to moderate. The role of MRI in the ongoing ProScreen trial is crucial and has a substantial impact on the screening process.Peer reviewe

Expected impact of MRI-related interreader variability on ProScreen prostate cancer screening trial: a pre-trial validation study

Background: The aim of this study is to investigate the potential impact of prostate magnetic resonance imaging (MRI) -related interreader variability on a population-based randomized prostate cancer screening trial (ProScreen). Methods: From January 2014 to January 2018, 100 men aged 50-63 years with clinical suspicion of prostate cancer (PCa) in Helsinki University Hospital underwent MRI. Nine radiologists individually reviewed the pseudonymized MRI scans of all 100 men in two ProScreen trial centers. All 100 men were biopsied according to a histological composite variable comprising radical prostatectomy histology (N = 38) or biopsy result within 1 year from the imaging (N = 62). Fleiss' kappa (kappa) was used to estimate the combined agreement between all individual radiologists. Sample data were subsequently extrapolated to 1000-men subgroups of the ProScreen cohort. Results: Altogether 89% men of the 100-men sample were diagnosed with PCa within a median of 2.4 years of follow-up. Clinically significant PCa (csPCa) was identified in 76% men. For all PCa, mean sensitivity was 79% (SD +/- 10%, range 62-96%), and mean specificity 60% (SD +/- 22%, range 27-82%). For csPCa (Gleason Grade 2-5) MRI was equally sensitive (mean 82%, SD +/- 9%, range 67-97%) but less specific (mean 47%, SD +/- 20%, range 21-75%). Interreader agreement for any lesion was fair (kappa 0.40) and for PI-RADS 4-5 lesions it was moderate (kappa 0.60). Upon extrapolating these data, the average sensitivity and specificity to a screening positive subgroup of 1000 men from ProScreen with a 30% prevalence of csPCa, 639 would be biopsied. Of these, 244 men would be true positive, and 395 false positive. Moreover, 361 men would not be referred to biopsy and among these, 56 csPCas would be missed. The variation among the radiologists was broad as the least sensitive radiologist would have twice as many men biopsied and almost three times more men would undergo unnecessary biopsies. Although the most sensitive radiologist would miss only 2.6% of csPCa (false negatives), the least sensitive radiologist would miss every third. Conclusions: Interreader agreement was fair to moderate. The role of MRI in the ongoing ProScreen trial is crucial and has a substantial impact on the screening process.Peer reviewe

Estimate of Opportunistic Prostate Specific Antigen Testing in the Finnish Randomized Study of Screening for Prostate Cancer

Purpose: Screening for prostate cancer remains controversial, although ERSPC (European Randomized Study of Screening for Prostate Cancer) showed a 21% relative reduction in prostate cancer mortality. The Finnish Randomized Study of Screening for Prostate Cancer, which is the largest component of ERSPC, demonstrated a statistically nonsignificant 16% mortality benefit in a separate analysis. The purpose of this study was to estimate the degree of contamination in the control arm of the Finnish trial. Materials and Methods: Altogether 48,295 and 31,872 men were randomized to the control and screening arms, respectively. The screening period was 1996 to 2007. The extent of prostate specific antigen testing was analyzed retrospectively using laboratory databases. The incidence of T1c prostate cancer (impalpable prostate cancer detected by elevated prostate specific antigen) was determined from the national Finnish Cancer Registry. Results: Approximately 1.4% of men had undergone prostate specific antigen testing 1 to 3 years before randomization. By the first 4, 8 and 12 years of follow-up 18.1%, 47.7% and 62.7% of men in the control arm had undergone prostate specific antigen testing at least once and in the screening arm the proportions were 69.8%, 81.1% and 85.2%, respectively. The cumulative incidence of T1c prostate cancer was 6.1% in the screening arm and 4.5% in the control arm (RR 1.21, 95% CI 1.13-1.30). Conclusions: A large proportion of men in the control arm had undergone a prostate specific antigen test during the 15-year followup. Contamination is likely to dilute differences in prostate cancer mortality between the arms in the Finnish screening trial.Peer reviewe

Estimating bias in causes of death ascertainment in the Finnish Randomized Study of Screening for Prostate Cancer

Background: Precise cause of death (CoD) ascertainment is crucial in any cancer screening trial to avoid bias from misclassification due to excessive recording of diagnosed cancer as a CoD in death certificates instead of non-cancer disease that actually caused death. We estimated whether there was bias in CoD determination between screening (SA) and control arms (CA) in a population-based prostate cancer (PCa) screening trial. Methods: Our trial is the largest component of the European Randomized Study of Screening for Prostate Cancer with more than 80,000 men. Randomly selected deaths in men with PCa (N = 442/2568 cases, 17.2%) were reviewed by an independent CoD committee. Median follow-up was 16.8 years in both arms. Results: Overdiagnosis of PCa was present in the SA as the risk ratio for PCa incidence was 1.19 (95% confidence interval (CI) 1.14-1.24). The hazard ratio (HR) for PCa mortality was 0.94 (95% CI 0.82-1.08) in favor of the SA. Agreement with official CoD registry was 94.6% (k = 0.88) in the SA and 95.4% (k = 0.91) in the CA. Altogether 14 PCa deaths were estimated as false-positive in both arms and exclusion of these resulted in HR 0.92 (95% CI 0.80-1.06). Conclusions: A small differential misclassification bias in ascertainment of CoD was present, most likely due to attribution bias (overdiagnosis in the SA). Maximum precision in CoD ascertainment can only be achieved with independent review of all deaths in the diseased population. However, this is cumbersome and expensive and may provide little benefit compared to random sampling. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe

Happamuuden aiheuttamat vesistöhaitat ja niiden torjuntakeinot Sanginjoella

Sanginjoki, Oulujoen alin sivujoki, on Oulun seudun tärkeimpiä virkistysalueita ja Merikosken kalatietä lähin potentiaalinen vaelluskalojen nousualue. Joen ajoittainen happamuus kuitenkin heikentää Sanginjoen virkistyskäytöllistä ja ekologista arvoa. Kaupunki ja vesi – Sanginjoen virkistyskäyttöarvon parantaminen ja ekologinen kunnostus (2008-2011) -hankkeessa selvitettiin Sanginjoen happamuuden alkuperää, seurattiin laajasti eri alueilta jokeen laskevien valumavesien pH:n muutoksia sekä testattiin menetelmiä happamien huuhtoumien ennaltaehkäisyyn ja neutralointiin. Tulosten perusteella laadittiin toimenpidesuunnitelma happamuuden ehkäisemiseksi sekä happamuuden aiheuttamien haittojen lieventämiseksi. Julkaisussa on myös esitetty tietoa Sanginjoen ja sen valuma-alueen ominaisuuksista, joen ekologisesta tilasta, vedenlaadun kehityksestä sekä happamuuden ehkäisemiseen soveltuvista menetelmistä. Tulosten perusteella Sanginjoen veden happamuus usein voimistuu virtaamien kasvaessa. Etenkin kesä- ja syyssateiden yhteydessä havaittiin alhaisia pH-lukemia, joihin vaikuttivat maaperä, kasvillisuus ja maankäyttö. Hapan huuhtouma on pääosin peräisin turvepitoisten maiden orgaanisesta huuhtoumasta, mutta paikallisesti vedenlaatuun voivat vaikuttaa alueella esiintyvät happamat sulfaattimaat ja mustaliuskealueet. Sanginjoen vesi on ollut myös luontaisesti hapanta lähinnä suo- ja turvemaiden happamien valumavesien johdosta, mutta happamuus on todennäköisesti lisääntynyt ihmistoiminnan vaikutuksesta. Sanginjoen valuma-alueella testattujen vesiensuojelu- ja kunnostusmenetelmien vaikutukset happamien valumavesien neutraloinnissa vaihtelivat, mutta osa menetelmistä osoittautui käyttökelpoiseksi ja niitä voidaan suositella käytettävän jatkossa niin Sanginjoella kuin vastaavilla happamuudesta kärsivillä kohteilla. Menetelmien kehittämistä ja erityisesti vaikutusten seurantaa tulee kuitenkin edelleen jatkaa. Hankkeessa testatut menetelmät ovat keinoja ihmistoiminnasta aiheutuvan happamuuden lisääntymisen torjunnassa. Parhaiten Sanginjoen ja muiden happamuudesta kärsivien vesistöjen hapanta kuormitusta ehkäistään huomioimalla maankäytössä happamuuden kannalta kriittisten turve- ja sulfidipitoisten alueiden ominaisuudet ja sijoittuminen jo ennen maankäytön toimenpiteitä ja kuormituksen syntymistä

A randomized trial of early detection of clinically significant prostate cancer (ProScreen) : study design and rationale

The current evidence of PSA-based prostate cancer screening shows a reduction in cause-specific mortality, but with substantial overdiagnosis. Recently, new developments in detection of clinically relevant prostate cancer include multiple kallikreins as biomarkers besides PSA, and multiparametric magnetic resonance imaging (mpMRI) for biopsy decision. They offer opportunities for improving the outcomes in screening, particularly reduction in overdiagnosis and higher specificity for potentially lethal cancer. A population-based randomized screening trial will be started, with 67,000 men aged 55-67 years at entry. A quarter of the men will be allocated to the intervention arm, and invited to screening. The control arm will receive no intervention. All men in the screening arm will be offered a serum PSA determination. Those with PSA of 3 ng/ml or higher will have an additional multi-kallikrein panel and those with indications of increased risk of clinically relevant prostate cancer will undergo mpMRI. Men with a malignancy-suspect finding in MRI are referred to targeted biopsies. Screening interval is 6 years for men with baseline PSA 3. The main outcome of the trial is prostate cancer mortality, with analysis at 10 and 15 years. The statistical power is sufficient for detecting a 28% reduction at 10 years and 22% at 15 years. The proposed study has the potential to provide the evidence to justify screening as a public health policy if mortality benefit can be sustained with substantially reduced overdiagnosis.Peer reviewe