Myoblast transplantation and adenoviral VEGF-C transfer in porcine model of coronary artery disease

Heart failure is a common and highly challenging medical disorder. The progressive increase of elderly population is expected to further reflect in heart failure incidence. Recent progress in cell transplantation therapy has provided a conceptual alternative for treatment of heart failure. Despite improved medical treatment and operative possibilities, end-stage coronary artery disease present a great medical challenge. It has been estimated that therapeutic angiogenesis would be the next major advance in the treatment of ischaemic heart disease. Gene transfer to augment neovascularization could be beneficial for such patients. We employed a porcine model to evaluate the angiogenic effect of vascular endothelial growth factor (VEGF)-C gene transfer. Ameroid-generated myocardial ischemia was produced and adenovirus encoding (ad)VEGF-C or β-galactosidase (LacZ) gene therapy was given intramyocardially during progressive coronary stenosis. Angiography, positron emission tomography (PET), single photon emission computed tomography (SPECT) and histology evidenced beneficial affects of the adVEGF-C gene transfer compared to adLacZ. The myocardial deterioration during progressive coronary stenosis seen in the control group was restrained in the treatment group. We observed an uneven occlusion rate of the coronary vessels with Ameroid constrictor. We developed a simple methodological improvement of Ameroid model by ligating of the Ameroid–stenosed coronary vessel. Improvement of the model was seen by a more reliable occlusion rate of the vessel concerned and a formation of a rather constant myocardial infarction. We assessed the spontaneous healing of the left ventricle (LV) in this new model by SPECT, PET, MRI, and angiography. Significant spontaneous improvement of myocardial perfusion and function was seen as well as diminishment of scar volume. Histologically more microvessels were seen in the border area of the lesion. Double staining of the myocytes in mitosis indicated more cardiomyocyte regeneration at the remote area of the lesion. The potential of autologous myoblast transplantation after ischaemia and infarction of porcine heart was evaluated. After ligation of stenosed coronary artery, autologous myoblast transplantation or control medium was directly injected into the myocardium at the lesion area. Assessed by MRI, improvement of diastolic function was seen in the myoblast-transplanted animals, but not in the control animals. Systolic function remained unchanged in both groups.

Phospholipid cytochrome c interactions

The ability of the peripherally associated membrane protein cytochrome c (cyt c) to bind phospholipids in vitro was studied using fluorescence spectroscopy and large unilamellar liposomes. Previous work has shown that cyt c can bind phospholipids using two distinct mecha- nisms and sites, the A-site and the C-site. This binding is mediated by electrostatic or hydrophobic interactions, respectively. Here, we focus on the mechanism underlying these interactions. A chemically modified cyt c mutant Nle91 was used to study the ATP-binding site, which is located near the evolutionarily invariant Arg 91 on the protein surface. This site was also demonstrated to mediate phospholipid binding, possibly by functioning as a phospholipid binding site. Circular dichroism spectroscopy, time resolved fluorescence spectroscopy of zinc- porphyrin modified [Zn2+-heme] cyt c and liposome binding studies of the Nle91 mutant were used to demonstrate that ATP induces a conformational change in membrane- bound cyt c. The ATP-induced conformational changes were mediated by Arg 91 and were most pronounced in cyt c bound to phospholipids via the C-site. It has been previously reported that the hydrophobic interaction between phospho- lipids and cyt c (C-site) includes the binding of a phospholipid acyl chain inside the protein. In this mechanism, which is known as extended phospholipid anchorage, the sn-2 acyl chain of a membrane phospholipid protrudes out of the membrane surface and is able to bind in a hydrophobic cavity in cyt c. Direct evidence for this type of bind- ing mechanism was obtained by studying cyt c/lipid interaction using fluorescent [Zn2+- heme] cyt c and fluorescence quenching of brominated fatty acids and phospholipids. Under certain conditions, cyt c can form fibrillar protein-lipid aggregates with neg- atively charged phospholipids. These aggregates resemble amyloid fibrils, which are involved in the pathogenesis of many diseases. Congo red staining of these fibers con- firmed the presence of amyloid structures. A set of phospholipid-binding proteins was also found to form similar aggregates, suggesting that phospholipid-induced amyloid formation could be a general mechanism of amyloidogenesis.

Adhesion,Presence and Antifouling of Deinococcus geothermalis in Paper Machine Environment

This thesis has two items: biofouling and antifouling in paper industry. Biofouling means unwanted microbial accumulation on surfaces causing e.g. disturbances in industrial processes, contamination of medical devices or of water distribution networks. Antifouling focuses on preventing accumulation of the biofilms in undesired places. Deinococcus geothermalis is a pink-pigmented, thermophilic bacterium, and extremely resistant towards radiation, UV-light and desiccation and known as a biofouler of paper machines forming firm and biocide resistant biofilms on the stainless steel surfaces. The compact structure of biofilm microcolonies of D. geothermalis E50051 and the adhesion into abiotic surfaces were investigated by confocal laser scanning microscope combined with carbohydrate specific fluorescently labelled lectins. The extracellular polymeric substance in D. geothermalis microcolonies was found to be a composite of at least five different glycoconjugates contributing to adhesion, functioning as structural elements, putative storages for water, gliding motility and likely also to protection. The adhesion threads that D. geothermalis seems to use to adhere on an abiotic surface and to anchor itself to the neighbouring cells were shown to be protein. Four protein components of type IV pilin were identified. In addition, the lectin staining showed that the adhesion threads were covered with galactose containing glycoconjugates. The threads were not exposed on planktic cells indicating their primary role in adhesion and in biofilm formation. I investigated by quantitative real-time PCR the presence of D. geothermalis in biofilms, deposits, process waters and paper end products from 24 paper and board mills. The primers designed for doing this were targeted to the 16S rRNA gene of D. geothermalis. We found D. geothermalis DNA from 9 machines, in total 16 samples of the 120 mill samples searched for. The total bacterial content varied in those samples between 107 to 3 ×1010 16S rRNA gene copies g-1. The proportion of D. geothermalis in those same samples was minor, 0.03 1.3 % of the total bacterial content. Nevertheless D. geothermalis may endanger paper quality as its DNA was shown in an end product. As an antifouling method towards biofilms we studied the electrochemical polarization. Two novel instruments were designed for this work. The double biofilm analyzer was designed for search for a polarization program that would eradicate D. geothermalis biofilm or from stainless steel under conditions simulating paper mill environment. The Radbox instrument was designed to study the generation of reactive oxygen species during the polarization that was effective in antifouling of D. geothermalis. We found that cathodic character and a pulsed mode of polarization were required to achieve detaching D. geothermalis biofilm from stainless steel. We also found that the efficiency of polarization was good on submerged, and poor on splash area biofilms. By adding oxidative biocides, bromochloro-5,5-dimethylhydantoin, 2,2-dibromo-2-cyanodiacetamide or peracetic acid gave additive value with polarization, being active on splash area biofilms. We showed that the cathodically weighted pulsed polarization that was active in removing D. geothermalis was also effective in generation of reactive oxygen species. It is possible that the antifouling effect relied on the generation of ROS on the polarized steel surfaces. Antifouling method successful towards D. geothermalis that is a tenacious biofouler and possesses a high tolerance to oxidative stressors could be functional also towards other biofoulers and applicable in wet industrial processes elsewhere.

Identification of molecules relevant for the invasiveness of fibrosarcomas and melanomas

Cancer is becoming the leading cause of deaths in the world. As 90% of all deaths from cancer are caused by metastasis, discovery of the mechanisms behind cancer cell invasion and metastasis is of utmost importance. Only new effective therapies targeting cancer progression can reduce cancer mortality rates. The aim of this study was to identify molecules that are relevant for tumor cell invasion and spreading in fibrosarcomas and melanomas, and to analyze their potential for cancer biomarkers or therapeutic targets. First, the gene expression changes of normal cells and transformed cells showing high invasiveness, S-adenosylmethionine decarboxylase (AdoMetDC)-transfected murine fibroblasts and human melanoma cells, were studied by microarray analyses. The function of the identified candidate molecules were then studied in detail in these cell lines. Finally, the physiological relevance of the identified changes was studied by immunohistochemical analyses of human sarcoma and melanoma specimens or by a mouse xenograft model. In fibrosarcoma cells, the most remarkable change detected was a dramatic up-regulation of the actin-sequestering molecule thymosin beta 4 (TB4), which was shown to be important for the transformed phenotype of the AdoMetDC-transfected cells (Amdc-s and -as). A sponge toxin latrunculin A, inhibiting the binding of TB4 to actin, was found to selectively inhibit the migration and invasion of these cells. Further, Amdc-s-induced mouse tumors and human high-grade sarcomas were found to show intense TB4 immunostaining. In addition to TB4, integrin subunits alfa 6 and beta 7 (ItgA6 and ItgB7) were found to be up-regulated in Amdc-s and -as cells. ItgA6 was shown to dimerize mainly with ItgB1 in Amdc-s. Inhibition of ItgA6 or ItgB1 function with neutralizing antibodies fully blocked the invasiveness of Amdc-s cells, and importantly also human HT-1080 fibrosarcoma cells, in three-dimensional (3D)-Matrigel mimicking tumor extracellular matrix (ECM). By immunohistochemical analyses, strong staining for ITGA6 was detected in human high-grade fibrosarcomas and other sarcomas, especially at the invasion fronts of the tumors. In the studied melanoma cell lines, the expression levels of the adhesion-related ECM proteins tenascin-C (TN-C), fibronectin (FN), and transforming growth factor beta-induced (TGFBI) were found to be highly up-regulated. By immunohistochemistry, intense TN-C and FN staining was detected in invasive and metastatic melanoma tumors, showing co-localization (together with procollagen-I) in tubular meshworks and channels around the invading melanoma cells. In vitro, TN-C and FN were further found to directly stimulate the migration of melanoma cells in 3D-collagen-I matrix. The third candidate protein, TGFBI, was found to be an anti-adhesive molecule for melanoma cells, and knockdown of its expression in metastatic melanoma cells (TGFBI-KD cells) led to dramatically impaired tumor growth in immunocompromized mice. Interestingly, the control tumors showed intense TGFBI immunostaining in the invasion fronts, showing partial co-localization with the fibrillar FN staining, whereas the small TGFBI-KD cell-induced tumors displayed amorphous, non-fibrillar FN staining. These data suggest an important role for TGFBI in FN fibrillogenesis and melanoma progression. In conclusion, we have identified several invasion-related molecules, which show potential for cancer diagnostic or prognostic markers, or therapeutic targets. Based on our previous and present fibrosarcoma studies, we propose the possibility of using ITGA6 antagonists (affecting tumor cell adhesion) in combination with TB4 inhibitors (affecting tumor cell migration) and cathepsin L inhibitors (affecting the degradation of basement membrane and ECM proteins) for the treatment of fibrosarcomas and other tumors overexpressing these molecules. With melanoma cells, in turn, we point to the importance of three secreted ECM proteins, TN-C, FN, and TGFBI, in melanoma progression. Of these, especially the potential of TN-C as a prognostic melanoma biomarker and TGFBI as a promising therapeutic target molecule are clearly worth additional studies.

The effects of organic environmental toxicants on hard tissue formation in developing tooth : An in vitro study in mice

Dioxins are organic toxicants that are known to impair tooth development, especially dental hard tissue formation. The most toxic dioxin congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further, clinical studies suggest that maternal smoking during pregnancy can affect child s tooth development. One of the main components of tobacco smoke is the group of non-halogenated polycyclic aromatic hydrocarbons (PAHs), a representative of which is 7,12-dimethylbenz[a]anthracene (DMBA). Tributyltin (TBT), an organic tin compound, has been shown to impair bone mineralization in experimental animals. In addition to exposure to organic toxicants, a well-established cause for enamel hypomineralization is excess fluoride intake. The principal aim of this thesis project was to examine in vitro if, in addition to dioxins, other organic environmental toxicants, like PAHs and organic tin compounds, have adverse effects on tooth development, specifically on formation and mineralization of the major dental hard tissues, the dentin and the enamel. The second aim was to investigate in vitro if fluoride could intensify the manifestation of the detrimental developmental dental effects elicited by TCDD. The study was conducted by culturing mandibular first and second molar tooth germs of E18 NMRI mouse embryos in a Trowell-type organ culture and exposing them to DMBA, TBT, and sodium fluoride (NaF) and/or TCDD at various concentrations during the secretory and mineralization stages of development. Specific methods used were HE-staining for studying cell and tissue morphology, BrdU-staining for cell proliferation, TUNEL-staining for apoptosis, and QPCR, in situ hybridization and immunohistochemistry for the expressions of selected genes associated with mineralization. This thesis work showed that DMBA, TBT, TCDD and NaF interfere with dentin and enamel formation of embryonic mouse tooth in vitro, and that fluoride can potentiate the harmful effect of TCDD. The results suggested that adverse effects of TBT involve altered expression of genes associated with mineralization, and that DMBA and TBT as well as NaF and TCDD together primarily affect dentin mineralization. Since amelogenesis does not start until mineralization of dentin begins, impaired enamel matrix secretion could be a secondary effect. Dioxins, PAHs and organotins are all liposoluble and can be transferred to the infant by breast-feeding. Since doses are usually very low, developmental toxicity on most of the organs is difficult to indentify clinically. However, tooth may act as an indicator of exposure, since the major dental hard tissues, the dentin and the enamel, are not replaced once they have been formed. Thus, disturbed dental hard tissue formation raises the question of more extensive developmental toxicity.

Molecular features of colorectal cancer predisposition and progression

Both inherited genetic variations and somatically acquired mutations drive cancer development. The aim of this thesis was to gain insight into the molecular mechanisms underlying colorectal cancer (CRC) predisposition and tumor progression. Whereas one-third of CRC may develop in the context of hereditary predisposition, the known highly penetrant syndromes only explain a small fraction of all cases. Genome-wide association studies have shown that ten common single nucleotide polymorphisms (SNPs) modestly predispose to CRC. Our population-based sample series of around thousand CRC cases and healthy controls was genotyped for these SNPs. Tumors of heterozygous patients were analyzed for allelic imbalance, in an attempt to reveal the role of these SNPs in somatic tumor progression. The risk allele of rs6983267 at 8q24 was favored in the tumors significantly more often than the neutral allele, indicating that this germline variant is somatically selected for. No imbalance targeting the risk allele was observed in the remaining loci, suggesting that most of the low-penetrance CRC SNPs mainly play a role in the early stages of the neoplastic process. The ten SNPs were further analyzed in 788 CRC cases, 97 of which had a family history of CRC, to evaluate their combined contribution. A significant association appeared between the overall number of risk alleles and familial CRC and these ten SNPs seem to explain around 9% of the familial clustering of CRC. Finding more CRC susceptibility alleles may facilitate individualized risk prediction and cancer prevention in the future. Microsatellite instability (MSI), resulting from defective mismatch repair function, is a hallmark of Lynch syndrome and observed in a subset of all CRCs. Our aim was to identify microsatellite frameshift mutations that inactivate tumor suppressor genes in MSI CRCs. By sequencing microsatellite repeats of underexpressed genes we found six novel MSI target genes that were frequently mutated in 100 MSI CRCs: 51% in GLYR1, 47% in ABCC5, 43% in WDTC1, 33% in ROCK1, 30% in OR51E2, and 28% in TCEB3. Immunohistochemical staining of GLYR1 revealed defective protein expression in homozygously mutated tumors, providing further support for the loss of function hypothesis. Another mutation screening effort sought to identify MSI target genes with putative oncogenic functions. Microsatellites were similarly sequenced in genes that were overexpressed and, upon mutation, predicted to avoid nonsense-mediated mRNA decay. The mitotic checkpoint kinase TTK harbored protein-elongating mutations in 59% of MSI CRCs and the mutant protein was detected in heterozygous MSI CRC cells. No checkpoint dysregulation or defective protein localization was observable however, and the biological relevance of this mutation may hence be related to other mechanisms. In conclusion, these two large-scale and unbiased efforts identified frequently mutated genes that are likely to contribute to the development of this cancer type and may be utilized in developing diagnostic and therapeutic applications.

Macrophages in regressed and progressed uveal melanoma

Uveal melanoma (UM) is the most common primary ocular malignancy in adults. In Finland, approximately 50 new cases are diagnosed yearly. Up to 50% of UM metastasize, mostly to the liver, although other organs are also affected. Despite improvements in the management of the primary tumour, the survival rates of patients with metastatic UM are poor. Until the 1970s, UMs were treated by enucleation i.e. removal of the eye. Currently, UM is usually treated by brachytherapy, which is known to influence tumour cells and blood vessels. UMs enucleated both primarily and secondarily after brachytherapy contain tumour-infiltrating macrophages, and a high number of macrophages in primary UM is associated with a shorter survival and a higher microvascular density (MVD) within the tumour tissue. The latter is independently associated with a shorter time to metastatic death. Macrophages have several diverse roles depending on their response to variable signals from the surrounding microenvironment. They function as scavengers, as producers of angiogenic and growth factors as well as proteases, which modulate extracellular matrix. Thus, tumour invasiveness and the risk for metastasis increase with increasing macrophage density. The aim of this study was to evaluate the effects of regression and progression of UM on macrophage numbers and microcirculation factors. Tumour regression is induced by primary brachytherapy, and tumour progression is evidenced by the development of metastases. Understanding the biological behaviour of UMs in the both states may help us in finding new treatment modalities against this disease. To achieve these aims case-control analyses of irradiated UMs and primarily-enucleated eyes (34 matched pairs) were performed. UMs were stained immunohistochemically to detect macrophages, extravascular matrix (EVM) loops and networks, and MVD. Following brachytherapy, a lower MVD was observed. The average number of macrophages remained unchanged. Considering that irradiated melanomas may still contain proliferating tumour cells, a clinically-relevant consequence of my study would be the reassurance that the risk for metastasis is likely to be reduced, given that the low MVD in untreated UMs indicates a favourable prognosis. The effect of progression on macrophages was studied in a paired analysis of primarily-enucleated UM and their corresponding hepatic metastases (48 pairs). A cross-sectional histopathological analysis of these pairs was carried out by staining both specimens in a similar way to the first study. MVD was greater in hepatic metastases than in corresponding primary tumours, and the survival of the patient tended to be shorter if hepatic metastases had a higher MVD. Hepatic metastases had also more dendritic macrophages than the primary UMs. Thus, the progression to metastasis seems to alter the inflammatory status within the tumour. Furthermore, determining MVD of biopsied hepatic metastases may serve as a supplementary tool in estimating the prognosis of patients with metastatic uveal melanoma. After irradiation, the majority of treated eyes have been clinically observed to have pigmented episcleral deposits. A noncomparative clinical case series of 211 irradiated UM eyes were studied by recording the number and location of pigmented episcleral deposits during follow-up visits after brachytherapy. For the first time, the study described pigmented episcleral deposits, which are found in the most UM eyes after brachytherapy, and proved them to consist of macrophages full with engulfed melanin particles. This knowledge may save patients from unnecessary enucleation, because episcleral pigmented deposits might be mistaken for extrascleral tumour growth. The presence of pigmented macrophage-related episcleral deposits was associated with plaque size and isotope rather than with tumour size, suggesting that, in addition to tumour regression, radiation atrophy of retinal pigment epithelium and choroid contributes to the formation of the deposits. In the paired (the same 34 pairs as in the first study) cross-sectional study of irradiated and non-irradiated UMs, clinically-visible episcleral deposits and migrating macrophages in other extratumoral tissues were studied histopathologically. Resident macrophages were present in extratumoral tissues in eyes with both irradiated and non-irradiated UM. Irradiation increased both the number of CD68+ macrophages in the sclera beneath the tumour and the number of clinically-observed episcleral macrophages aggregates. Brachytherapy seemed to alter the route of migration of macrophages: after irradiation, macrophages migrated preferentially through the sclera while in non-irradiated UMs they seemed to migrate more along the choroid. In order to understand the influence of these routes on tumour progression and regression in the future, labelling and tracking of activated macrophages in vivo is required.

Cox-2, tenascin, CRP, and ingraft chimerism in a model of post-transplant obliterative bronchiolitis

Chronic rejection in the form of obliterative bronchiolitis (OB) is the major cause of death 5 years after lung transplantation. The exact mechanism of OB remains unclear. This study focused on the role of cyclo-oxygenase (COX) -2, tenascin, and C-reactive protein (CRP) expression, and the occurrence of ingraft chimerism (= cells from two genetically distinct individuals in a same individual) in post-transplant OB development. In our porcine model, OB developed invariably in allografts, while autografts stayed patent. The histological changes were similar to those seen in human OB. In order to delay or prevent obliteration, animals were medicated according to certain protocol. In the beginning of the bronchial allograft reaction, COX-2 induction occurred in airway epithelial cells prior to luminal obliteration. COX-2 expression in macrophages and fibroblasts paralleled the onset of inflammation and fibroblast proliferation. This study demonstrated for the first time, that COX-2 expression is associated with the early stage of post- transplant obliterative airway disease. Tenascin expression in the respiratory epithelium appeared to be predictive of histologic features observed in human OB, and influx of immune cells. Expression in the bronchial wall and in the early obliterative lesions coincided with the onset of onset of fibroblast and inflammatory cell proliferation in the early stage of OB and was predictive of further influx of inflammatory and immune cells. CRP expression in the bronchial wall coincided with the remodelling process. High grade of bronchial wall CRP staining intensity predicted inflammation, accelerated fibroproliferation, and luminal obliteration, which are all features of OB. In the early obliterative plaque, majority of cells expressed CRP, but in mature, collagen-rich plaque, expression declined. Local CRP expression might be a response to inflammation and it might promote the development of OB. Early appearance of chimeric (= recipient-derived) cells in the graft airway epithelium predicted epithelial cell injury and obliteration of the bronchial lumen, which both are features of OB. Chimeric cells appeared in the airway epithelium after repair following transplantation-induced ischemic injury. Ingraft chimerism might be a mechanism to repair alloimmune-mediated tissue injury and to protect allografts from rejection after transplantation. The results of this study indicate, that COX-2, tenascin, CRP, and ingraft chimerism have a role in OB development. These findings increase the understanding of the mechanisms of OB, which may be beneficial in further development of diagnostic options.

Role of GDNF and its Cross-Talk with Other Growth Factors in the Dopaminergic System

Parkinson´s Disease (PD) is a neurodegenerative movement disorder resulting from loss of dopaminergic (DA) neurons in substantia nigra (SN). Possible causative treatment strategies for PD include neurotrophic factors, which protect and in some cases restore the function of dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors have been to date the most promising candidates for treatment of PD, demonstrating both neuroprotective and neurorestorative properties. We have investigated the role of GDNF in the rodent dopaminergic system and its possible crosstalk with other growth factors. We characterized the GDNF-induced gene expression changes by DNA microarray analysis in different neuronal systems, including in vitro cultured Neuro2A cells treated with GDNF, as well as midbrains from GDNF heterozygous (Hz) knockout mice. These microarray experiments, resulted in the identification of GDNF-induced genes, which were also confirmed by other methods. Further analysis of the dopaminergic system of GDNF Hz mice demonstrated about 40% reduction in GDNF levels, revealed increased intracellular dopamine concentrations and FosB/DeltaFosB expression in striatal areas. These animals did not show any significant changes in behavioural analysis of acute and repeated cocaine administration on locomotor activity, nor did they exhibit any changes in dopamine output following treatment with acute cocaine. We further analysed the significance of GDNF receptor RET signalling in dopaminergic system of MEN2B knock-in animals with constitutively active Ret. The MEN2B animals showed a robust increase in extracellular dopamine and its metabolite levels in striatum, increased tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels by immunohistochemical staining and Western blotting, as well as increased Th mRNA levels in SN. MEN2B mice had increased number of DA neurons in SN by about 25% and they also exhibited increased sensitivity to the stimulatory effects of cocaine. We also developed a semi-throughput in vitro micro-island assay for the quantification of neuronal survival and TH levels by computer-assisted methodology from limited amounts of tissue. This assay can be applied for the initial screening for dopaminotrophic molecules, as well as chemical drug library screening. It is applicable to any neuronal system for the screening of neurotrophic molecules. Since our microarray experiments revealed possible GDNF-VEGF-C crosstalk we further concentrated on studying the neurotrophic effects of VEGF-C. We showed that VEGF-C acts as a neurotrophic molecule for the DA neurons both in vitro and in vivo, however without additive effect when used together with GDNF. The neuroprotective effect for VEGF-C in vivo in rat 6-OHDA model of PD was demonstrated. The possible signalling mechanisms of VEGF-C in the nervous system were investigated - infusion of VEGF-C to rat brain induced ERK activation, however no direct activation of RET signalling in vitro was found. VEGF-C treatment of rat striatum lead to up-regulation of VEGFR-1-3, indicating that VEGF-C can regulate the expression level of its own receptor. VEGF-C dopaminotrophic activity in vivo was further supported by increased vascular tissue in the neuroprotection experiments.