Browsing by Subject "angiogenesis"

Sort by: Order: Results:

Now showing items 1-20 of 23
  • Rautiainen, Swarna (Helsingin yliopisto, 2020)
    Endothelial dysfunction is a common characteristic of several diseases including diabetes mellitus, coronary heart disease and stroke. Healthy endothelium ensures vascular homeostasis, regulation of blood flow and the exchange of oxygen and nutrients, as well as immune cell filtration to the surrounding tissues. In many cases, endothelial dysfunction results in ischemia in the surrounding tissues impairing cellular regeneration mechanisms, which can lead to tissue necrosis in the worst case. Therapeutic angiogenesis via stem cell transplantation aims to restore tissue blood flow and thus aid in tissue regeneration and restoration of a functioning tissue. Adipose derived stem/stromal cells (ASC) are a stem cell population with a multilineage differentiation ability. They have been shown to differentiate towards adipogenic, osteogenic, chondrogenic, myogenic and neurogenic lineages among others. Their easy obtainability from liposuction material and abundance in the adipose tissue makes them an especially practical and favorable cell option for stem cell research. In angiogenesis research, ASCs are commonly used in a co-culture with an endothelial cell (EC) type such as human umbilical vein endothelial cell. ASCs secrete extracellular vesicles (EV) that are small membrane bound vesicles with a diameter ranging from 40-1000 nm, and which have the ability to alter the behavior of target cells through their cargo. EV cargo consists of microRNAs, messenger-RNAs and proteins, and the EV cargo of ASCs has been shown to have proangiogenic effects. The aim of this work was to review what is currently known about ASC ability to promote angiogenesis through paracrine secretion and differentiation into endothelial cells or pericytes, interactions between ASCs and endothelial cells in the angiogenesis promoting process and the role of ASC extracellular vesicles in promoting angiogenesis. The methods for this work were database research of related articles using scientific databases and search engines, article categorization and reading, and finally manuscript production. It can be concluded from the current literature that a co-culture environment of ASCs and an endothelial cell type supports the formation of tube-like structures in vitro. Additional insulin like growth factor 1 in culture medium enhances the expression of angiogenesis-related growth factors in both cell types via PI3K/AKT signaling pathway. Further, the activation of platelet derived growth factor receptor β supports ASC ability to promote vascular network formation. On the contrary, the presence of ASC secreted activin A results in the inhibition of vascular network formation. ASCs can differentiate into endothelial cells particularly in three-dimensional culture conditions. In addition, fibroblast growth factor 2 and the activation of the AKT-pathway are crucial for endothelial differentiation. In addition, ASCs have the ability to differentiate into pericytes and assume a stabilizing role on the outside of the microvessels. Concerning ASC derived EVs and their cargo, miR-31, miR-125a and miR-126 have proangiogenic effects in vitro and in vivo. Proangiogenic miRNAs in ASC EV cargo are miR-181b-5p and the let7-family, out of which miR-181b-5p upregulates vascular endothelial growth factor and hypoxia-inducible factor 1α and let7-family influences tube formation ability of ECs. In vivo, ASC derived EVs support fat grafting, enhance wound healing both in healthy and diabetic environment, and provide cardioprotection. Therefore, ASC EVs show potential for therapeutic angiogenesis but currently there is a lack of clinical trials in EV research.
  • Kallio, Pauliina; Jokinen, Elina; Das, Suvendu; Högström, Jenny; Heino, Sarika; Lähde, Marianne; Alitalo, Kari (Helsingin yliopisto, 2018)
    Radiation induced tumor cell death is strongly dependent on oxygen. As abnormal tumor vasculature promotes tumor hypoxia, drugs that induce vascular normalization, such as the anti-vascular endothelial growth factor (VEGF) antibody, have been tested as radiation sensitizers in preclinical and clinical settings. The insufficient benefit obtained with anti-VEGF therapy prompted us to test if antibodies blocking the endothelial growth factor angiopoietin-2 (Ang2) could improve the effect of radiation in mouse tumor allografts and human tumor xenografts. Mouse or human tumor cells were injected subcutaneously in isogenic immunocompetent or immunodeficient (NSG) mice, respectively, and tumors were allowed to form. The mice were injected with anti-Ang2 or control antibodies every three or four days starting three days before 3x2 Gy or 4x0.5 Gy whole-body radiation, followed by analysis of tumor growth, histology, vasculature, hypoxia and necrosis. Combination treatment with anti-Ang2 and radiation improved tumor growth inhibition and extended the survival of mice with melanoma or colorectal carcinoma allografts. A similar anti-Ang2 plus radiation response was also obtained in immunodeficient mice implanted with a human colorectal carcinoma xenograft, indicating that the adaptive immune response was not essential for the effect. Histological and immunohistochemical analysis of the tumors showed that the combination treatment decreased tumor vasculature, and increased tumor hypoxia and tumor necrosis in comparison with control tumors and tumors treated with the monotherapies. Our results suggest that a combination of Ang2 blocking antibodies and radiation increases tumor growth inhibition and extends the survival of tumor-bearing mice. Significance: These findings offer a preclinical rationale for further testing of the use of Ang2 blocking antibodies in combination with radiation to improve the overall outcome of cancer treatment.
  • Heiskanen, Suvi (Helsingfors universitet, 2010)
    Cyclin dependent kinase 5 (Cdk5) is studied to take part in the migration neurons and development of brain. It is proven to participate also in the mediation of endothelial cell migration and angiogenesis. Angiogenesis is a crucial physiological mechanism in mediating wound healing and menstrual cycle among other functions. It is also important in some patophysiological processes like diabetic retinopathy and tumour outgrow. Tumour is shown to need its own vascularisation after reaching a size of 2-3 mm as a diameter in order to proceed growing. This makes Cdk5 a potential therapeutic target in regulating angiogenesis. In order to be activated, Cdk5 forms a complex together with its neuronal activator p35 or p39, or with their respective cleavage products p25 or p29. The mechanisms, how Cdk5 is activated in human ehdothelial cells has not been studied before. This master thesis is to evaluate the existence of Cdk5 activators p35 and p39 and their respective cleavage products in spreading human umbilical vein endothelial cells (HUVECs) to mimic the cell migration by freshly plating the cells. In our studies we performed western blot analysis and quantitative PCR analysis to investigate the expression of p35 and p25 in spreading HUVECs in different time points. We also performed an immunofluorescence assay to investigate the localisation of p35 and p25 in spreading HUVECs using confocal laser scanning microscopy (CLSM). The expression of p35 and p25 was also studied after growth factor stimulation (VEGF, FGF). The expression of the activator p39 in spreading HUVECs was studied using quantitative PCR method. Finally we investigated the interaction of Cdk5 with its activator p35 and its cleavage product p25 in spreading HUVECs using immunoprecipitation (IP). We were able to show in our studies that the activators p35 and p25 are expressed in HUVECs and that their expression is changing in spreading HUVECs in different time points. Additionally we were able to show, that p35 and p25 are partly localized in periphery in spreading cells. We were able to show that also the activator p39 is expressed in spreading HUVECs, but its relative amount was shown to be only a small portion of p35 in HUVECs. We were able to prove the interaction of Cdk5 with its activator p35 and p25 using immunoprecipitation, although the result could not be completely verified. Stimulation with growth factors showed no appreciable changes in the expression of p35 or p25. Based on the results, we can state that both the activator p35 and p39, and at least p25, the cleavage product p35, are expressed also in HUVECs. As they are neuronal activators of Cdk5 and Cdk5 has shown to participate in the mediation of angiogenesis and endothelial cell migration, the results amplify our hypothesis that also these activators might have a role in mediating endothelial cell migration and angiogenesis. Nevertheless to assure it, to specify the possible different roles of each activators and their interaction with Cdk5, further studies are needed.
  • Kivelä, Riikka; Hemanthakumar, Karthik Amudhala; Vaparanta, Katri; Robciuc, Marius; Izumiya, Yasuhiro; Kidoya, Hiroyasu; Takakura, Nobuyuki; Peng, Xuyang; Sawyer, Douglas B.; Elenius, Klaus; Walsh, Kenneth; Alitalo, Kari (2019)
    Background: Heart failure, which is a major global health problem, is often preceded by pathological cardiac hypertrophy. The expansion of the cardiac vasculature, to maintain adequate supply of oxygen and nutrients, is a key determinant of whether the heart grows in a physiological compensated manner or a pathological decompensated manner. Bidirectional endothelial cell (EC)-cardiomyocyte (CMC) cross talk via cardiokine and angiocrine signaling plays an essential role in the regulation of cardiac growth and homeostasis. Currently, the mechanisms involved in the EC-CMC interaction are not fully understood, and very little is known about the EC-derived signals involved. Understanding how an excess of angiogenesis induces cardiac hypertrophy and how ECs regulate CMC homeostasis could provide novel therapeutic targets for heart failure. Methods: Genetic mouse models were used to delete vascular endothelial growth factor (VEGF) receptors, adeno-associated viral vectors to transduce the myocardium, and pharmacological inhibitors to block VEGF and ErbB signaling in vivo. Cell culture experiments were used for mechanistic studies, and quantitative polymerase chain reaction, microarrays, ELISA, and immunohistochemistry were used to analyze the cardiac phenotypes. Results: Both EC deletion of VEGF receptor (VEGFR)-1 and adeno-associated viral vector-mediated delivery of the VEGFR1-specific ligands VEGF-B or placental growth factor into the myocardium increased the coronary vasculature and induced CMC hypertrophy in adult mice. The resulting cardiac hypertrophy was physiological, as indicated by preserved cardiac function and exercise capacity and lack of pathological gene activation. These changes were mediated by increased VEGF signaling via endothelial VEGFR2, because the effects of VEGF-B and placental growth factor on both angiogenesis and CMC growth were fully inhibited by treatment with antibodies blocking VEGFR2 or by endothelial deletion of VEGFR2. To identify activated pathways downstream of VEGFR2, whole-genome transcriptomics and secretome analyses were performed, and the Notch and ErbB pathways were shown to be involved in transducing signals for EC-CMC cross talk in response to angiogenesis. Pharmacological or genetic blocking of ErbB signaling also inhibited part of the VEGF-B-induced effects in the heart. Conclusions: This study reveals that cross talk between the EC VEGFR2 and CMC ErbB signaling pathways coordinates CMC hypertrophy with angiogenesis, contributing to physiological cardiac growth.
  • de Andrade, A. L. D. L.; de Oliveira, C. E.; Dourado, M. R.; Macedo, C. C. S.; Winck, F. V.; Paes Leme, A. F.; Salo, T.; Coletta, R. D.; de Almeida Freitas, R.; Galvao, H. C. (2018)
    BackgroundA new intercellular communication mode established by neoplastic cells and tumor microenvironment components is based on extracellular vesicles (EVs). However, the biological effects of the EVs released by tumor cells on angiogenesis are not completely understood. Here, we aimed to understand the biological effects of EVs isolated from two cell lines of oral squamous cell carcinoma (OSCC) (SCC15 and HSC3) on endothelial cell tubulogenesis. MethodsOSCC-derived EVs were isolated with a polymer-based precipitation method, quantified using nanoparticle tracking analysis and verified for EV markers by dot blot. Functional assays were performed to assess the angiogenic potential of the OSCC-derived EVs. ResultsThe results showed that EVs derived from both cell lines displayed typical spherical-shaped morphology and expressed the EV markers CD63 and Annexin II. Although the average particle concentration and size were quite similar, SCC15-derived EVs promoted a pronounced tubular formation associated with significant migration and apoptosis rates of the endothelial cells, whereas EVs derived from HSC3 cells inhibited significantly endothelial cell tubulogenesis and proliferation. ConclusionThe findings of this study reveal that EVs derived from different OSCC cell lines by a polymer-based precipitation method promote pro- or anti-angiogenic effects.
  • Jeltsch, Markku (University of Helsinki, 1997)
  • Taskinen, Juuso (Helsingin yliopisto, 2019)
    Human umbilical vein endothelial cells are responsible for maintaining and forming new vessels from existing ones, in a biological process called sprouting angiogenesis. Sprouting angiogenesis is a crucial mechanism for the resolution of hypoxia and normal development of tissues. It also plays a key role in internal plague hemorrhages, which can lead to embolisms and other cardiovascular complications. Angiogenesis is also crucial for cancer development. Sprouting angiogenesis is initiated by hypoxic tissue excreted vascular endothelial growth factor gradient, which induces normal endothelial cells into either a proliferative stalk cell or a signal sensing tip cell phenotype. Both of these cell types depend on the rapid flow of lipids to their plasma membrane, either to form plasma membrane protrusions in tip cells or as new plasma membrane material in dividing stalk cells. This flow is envisioned to involve both vesicle-mediated and non-vesicular mechanisms. A major non-vesicular route of lipid transfer occurs at membrane contact sites via lipid transport proteins. Furthermore, lipids can be transported to the plasma membrane by the direct fusion of vesicles or endosomes with the plasma membrane This thesis set out to explore the role of two membrane contact site proteins, oxysterol-binding protein- related protein 2 and protrudin, in angiogenesis and lipid transfer. Their role was examined by RNA-sequencing transient knock-down samples of these proteins in HUVECs. The RNA-sequencing data was examined by differential expression, gene ontology overrepresentation and gene set enrichment analyses. Gene expression analysis provided almost 10 000 significantly changed transcripts (adjusted p-values < 0.05), in each silenced cell type. The distribution of differentially expressed genes in oxysterol-binding protein- related protein 2 silenced cells, is skewed toward negative fold changes, whereas the distribution of differentially expressed genes in protrudin silenced samples is normally distributed. The results also show significant changes in gene ontologies related to proliferation, cell cycle, angiogenesis as well as hypoxia in both sample types. Gene set enrichment analysis showed upregulation in angiogenesis related pathways, such as the PI3K-Akt and MAPK pathways, in both samples. Significant downregulation was present in cell cycle related pathways and cholesterol biosynthesis pathway in both ORP2 and protrudin silenced samples.
  • Gebraad, Arjen; Ohlsbom, Roope; Miettinen, Juho J.; Emeh, Promise; Pakarinen, Toni-Karri; Manninen, Mikko; Eskelinen, Antti; Kuismanen, Kirsi; Slipicevic, Ana; Lehmann, Fredrik; Nupponen, Nina N.; Heckman, Caroline A.; Miettinen, Susanna (2022)
    Mesenchymal stem/stromal cells (MSCs) are self-renewing and multipotent progenitors, which constitute the main cellular compartment of the bone marrow stroma. Because MSCs have an important role in the pathogenesis of multiple myeloma, it is essential to know if novel drugs target MSCs. Melflufen is a novel anticancer peptide-drug conjugate compound for patients with relapsed refractory multiple myeloma. Here, we studied the cytotoxicity of melflufen, melphalan and doxorubicin in healthy human bone marrow-derived MSCs (BMSCs) and how these drugs affect BMSC proliferation. We established co-cultures of BMSCs with MM.1S myeloma cells to see if BMSCs increase or decrease the cytotoxicity of melflufen, melphalan, bortezomib and doxorubicin. We evaluated how the drugs affect BMSC differentiation into adipocytes and osteoblasts and the BMSC-supported formation of vascular networks. Our results showed that BMSCs were more sensitive to melflufen than to melphalan. The cytotoxicity of melflufen in myeloma cells was not affected by the co-culture with BMSCs, as was the case for melphalan, bortezomib and doxorubicin. Adipogenesis, osteogenesis and BMSC-mediated angiogenesis were all affected by melflufen. Melphalan and doxorubicin affected BMSC differentiation in similar ways. The effects on adipogenesis and osteogenesis were not solely because of effects on proliferation, seen from the differential expression of differentiation markers normalized by cell number. Overall, our results indicate that melflufen has a significant impact on BMSCs, which could possibly affect therapy outcome.
  • Almahmoudi, Rabeia; Salem, Abdelhakim; Murshid, Sakhr; Dourado, Mauricio Rocha; Apu, Ehsanul Hoque; Salo, Tuula; Al-Samadi, Ahmed (2019)
    We recently showed that extracellular interleukin-17F (IL-17F) correlates with better disease-specific survival in oral tongue squamous cell carcinoma (OTSCC) patients. However, the underlying mechanisms of such effect remain obscure. Here, we used qRT-PCR to assess the expression of IL-17F and its receptors (IL-17RA and IL-17RC) in two OTSCC cell lines (HSC-3 and SCC-25) and in normal human oral keratinocytes (HOKs). IL-17F effects on cancer cell proliferation, migration, and invasion were studied using a live-imaging IncuCyte system, and a Caspase-3/7 reagent was used for testing apoptosis. 3D tumor spheroids were utilized to assess the impact of IL-17F on invasion with or without cancer-associated fibroblasts (CAFs). Tube-formation assays were used to examine the effects of IL-17F on angiogenesis using human umbilical vein endothelial cells (HUVEC). OTSCC cells express low levels of IL-17F, IL-17RA, and IL-17RC mRNA compared with HOKs. IL-17F inhibited cell proliferation and random migration of highly invasive HSC-3 cells. CAFs promoted OTSCC invasion in tumor spheroids, whereas IL-17F eliminated such effect. IL-17F suppressed HUVEC tube formation in a dose-dependent manner. Collectively, we suggest that IL-17F counteracts the pro-tumorigenic activity in OTSCC. Due to its downregulation in tumor cells and inhibitory activity in in vitro cancer models, targeting IL-17F or its regulatory pathways could lead to promising immunotherapeutic strategies against OTSCC.
  • Jha, Sawan Kumar; Rauniyar, Khusbu; Chronowska, Ewa; Mattonet, Kenny; Maina, Eunice; Koistinen, Hannu; Stenman, Ulf Håkan; Alitalo, Kari; Jeltsch, Michael (2019)
    Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, e.g. in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D.
  • Gucciardo, Erika; Loukovaara, Sirpa; Salven, Petri; Lehti, Kaisa (2018)
    Diabetic retinopathy (DR) is the most common diabetic microvascular complication and major cause of blindness in working-age adults. According to the level of microvascular degeneration and ischemic damage, DR is classified into non-proliferative DR (NPDR), and end-stage, proliferative DR (PDR). Despite advances in the disease etiology and pathogenesis, molecular understanding of end-stage PDR, characterized by ischemia- and inflammation-associated neovascularization and fibrosis, remains incomplete due to the limited availability of ideal clinical samples and experimental research models. Since a great portion of patients do not benefit from current treatments, improved therapies are essential. DR is known to be a complex and multifactorial disease featuring the interplay of microvascular, neurodegenerative, metabolic, genetic/epigenetic, immunological, and inflammation-related factors. Particularly, deeper knowledge on the mechanisms and pathophysiology of most advanced PDR is critical. Lymphatic-like vessel formation coupled with abnormal endothelial differentiation and progenitor cell involvement in the neovascularization associated with PDR are novel recent findings which hold potential for improved DR treatment. Understanding the underlying mechanisms of PDR pathogenesis is therefore crucial. To this goal, multidisciplinary approaches and new ex vivo models have been developed for a more comprehensive molecular, cellular and tissue-level understanding of the disease. This is the first step to gain the needed information on how PDR can be better evaluated, stratified, and treated.
  • Paech, Jennifer Bianca (Helsingin yliopisto, 2020)
    Cardiovascular diseases are the leading cause of death globally. Especially pathological cardiac hypertrophy can be a trigger for severe pathological conditions, such as congestive heart failure. Previously, overexpression of vascular endothelial growth factor B (VEGF-B) in cardiomyocytes has been shown to lead to cardiac hypertrophy, but in a reversible, physiological way. Furthermore, VEGF-B overexpression leads to significant expansion of the coronary vascular tree. This study compares transcriptomics of postnatal and adult murine cardiac endothelial cells (ECs) and examines the transcriptional changes in response to VEGF-B transgene, plus the effect of the VEGF-B transgene on recovery of the murine cardiac ECs from myocardial infarction (MI). I analyzed isolated ECs from VEGF-B transgenic and AAV-VEGF-B transduced mice with single-cell RNA sequencing. The markers used for identification of the cell types applies to all experimental groups, although the proportions of cells differ among the conditions. The myocardial VEGF-B transgene promotes EC proliferation during development and boosts endothelial proliferation also in adult mice both in physiological conditions and after MI. Trajectory analysis indicates that ECs from the VEGF-B treated mice follow a distinct trajectory to enter the cell cycle after MI. These results suggest VEGF-B gene therapy as a new tool for coronary vessel remodeling, which could open new perspectives in the prevention and treatment of myocardial infarction.
  • Tonttila, Kialiina (Helsingin yliopisto, 2021)
    Respirometry is a polarographic method that provides insights into mitochondrial respiratory capacity – specifically to electron transport chain (ETC) complexes I to V –, mitochondrial integrity and energy metabolism. The limitation of the respiratory measurements has been that it requires freshly isolated mitochondria or tissue sample. Long-term preservation of mitochondrial function in frozen samples has been a considerable challenge, since the membrane integrity of the mitochondria is lost during the freezing process. Thus, samples do not display coupled respiration. However, previous studies have found that despite coupled respiration is impaired the individual ETC complexes and the ability of ETC supercomplexes to consume oxygen are not destroyed due to freezing and thawing. On the basis of this knowledge, recently published article presented a novel protocol that overcomes the damages caused by freeze-thaw cycles. The protocol also enables respiration measurement of ETC complexes I-IV by using Seahorse XF96 Extracellular flux analyzer. In this MSc thesis I modified and optimized the aforementioned protocol for Oroboros O2k high- resolution respirometry using frozen skeletal muscle samples. In addition, this study provides an optimized sample preparation protocol for frozen muscle samples and respiration measurement. The new method broadens the possibilities within mitochondrial respiration studies since Oroboros O2k high-resolution respirometry records results with high sensitivity without limiting the number of substrates used. The possibility to use frozen samples reduces research costs, simplifies logistics and enables retrospective studies with previously stored frozen tissue samples. I also utilized the optimized respiration measurement protocol to study metabolic effects of combined gene therapy in skeletal muscle. This gene therapy mimics the positive effects of exercise by inducing skeletal muscle growth and angiogenesis. The mimicking effect was induced by systemic delivery of adeno-associated viral vectors encoding pro-myostatin and VEGF-B. In previous studies inhibition of myostatin has been connected to compromised oxidative capacity and vascular rarefaction. In contrast, VEGF-B has demonstrated to induce angiogenesis in several tissues. Thus, my hypothesis was that combination gene therapy would result in better mitochondrial function than pro-myostatin alone. Results from this study indicate that moderate inhibition of myostatin signaling by pro-myostatin using rAAV vectors could provide enhancements in ETC function when it is induced independently or combined with rAAV-VEGF-B. This result lays a solid foundation for future research and could provide a new therapeutic option against muscle loss and related metabolic diseases.
  • Koponen, Annika; Pan, Guoping; Kivelä, Annukka M.; Ralko, Arthur; Taskinen, Juuso H.; Arora, Amita; Kosonen, Riikka; Kari, Otto K.; Ndika, Joseph; Ikonen, Elina; Cho, Wonhwa; Yan, Daoguang; Olkkonen, Vesa M. (2020)
    Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P(2)countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P(2)content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P(2)distribution, angiogenic signaling, and angiogenesis. We show that ORP2 knock-down modifies the distribution of cholesterol accessible to a D4H probe, between late endosomes and the PM. Depletion of ORP2 from ECs inhibits their angiogenic tube formation capacity, alters the gene expression of angiogenic signaling pathways such as VEGFR2, Akt, mTOR, eNOS, and Notch, and reduces EC migration, proliferation, and cell viability. We show that ORP2 regulates the integrity of VEGFR2 at the PM in a cholesterol-dependent manner, the depletion of ORP2 resulting in proteolytic cleavage by matrix metalloproteinases, and reduced activity of VEGFR2 and its downstream signaling. We demonstrate that ORP2 depletion increases the PM PI(4,5)P(2)coincident with altered F-actin morphology, and reduces both VEGFR2 and cholesterol in buoyant raft membranes. Moreover, ORP2 knock-down suppresses the expression of the lipid raft-associated proteins VE-cadherin and caveolin-1. Analysis of the retinal microvasculature in ORP2 knock-out mice generated during this study demonstrates the subtle alterations of morphology characterized by reduced vessel length and increased density of tip cells and perpendicular sprouts. Gene expression changes in the retina suggest disturbance of sterol homeostasis, downregulation of VE-cadherin, and a putative disturbance of Notch signaling. Our data identifies ORP2 as a novel regulator of EC cholesterol and PI(4,5)P(2)homeostasis and cholesterol-dependent angiogenic signaling.
  • Guryanov, Ivan; Tennikova, Tatiana; Urtti, Arto (2021)
    Vascular endothelial growth factors (VEGFs) are the family of extracellular signaling proteins involved in the processes of angiogenesis. VEGFA overexpression and altered regulation of VEGFA signaling pathways lead to pathological angiogenesis, which contributes to the progression of various diseases, such as age-related macular degeneration and cancer. Monoclonal antibodies and decoy receptors have been extensively used in the anti-angiogenic therapies for the neutralization of VEGFA. However, multiple side effects, solubility and aggregation issues, and the involvement of compensatory VEGFA-independent pro-angiogenic mechanisms limit the use of the existing VEGFA inhibitors. Short chemically synthesized VEGFA binding peptides are a promising alternative to these full-length proteins. In this review, we summarize anti-VEGFA peptides identified so far and discuss the molecular basis of their inhibitory activity to highlight their pharmacological potential as anti-angiogenic drugs.
  • Jokinen, Birgitta (Helsingfors universitet, 2010)
    Angiogenesis may be regarded as one of the most important phenomena involved in basic physiology as well as in numerous pathological conditions. Angiogenesis is a multistep process involving the balance of pro- and con-angiogenic factors. Several studies have suggested that angiogenesis is regulated in vitro and in vivo by peptides thymosin ȕ4 (Tȕ4) and tetrapeptide Ac-SDKP (N-acetyl-seryl-aspartyl-lysyl-proline). There are also studies supporting the view that Ac-SDKP, a peptide fragment is released from the proline-containing C-terminus of Tȕ4 (43-mer) by hydrolyzing prolyl oligopeptidase (POP). POP is a widely existing serine protease cleaving oligopeptides of no longer than 30 amino acids. Thus, Tȕ4 should first be cleaved into a shorter peptide by some other, yet unknown peptidase. POP has been mostly studied in memory and learning disorders as well as in neurodegenerative diseases. The true physiological character of POP is still unresolved. In this Master's thesis, the associations of the factors involved in angiogenesis are reviewed in the literature part as well as the character, presence and function of the angiogenic molecules 7ȕ4, Ac-SDKP and POP. In the experimental part attempts were made to find whether POP and Tȕ4 increase Ac-SDKP formation and capillary tube network and consequently, whether the POP activity, tetrapeptide and capillary formation could be inhibited by the proline-spesific POP inhibitor KYP-2047. The study had two phases. The first phase included POP activity and Ac-SDKP measurements(time period 0-180 min) with Wistar rat kidney homogenates. Study groups were 0,1 and 0,5 µM KYP-2047 (+2 µM Tȕ4), 1:20 (0.625 µM) human recombinant POP (+ 2 µM Tȕ4), 2 µM 7ȕ4 (pos. control) and raw homogenate (neg. control). The second phase involved the study of capillary formation (time period 0-180 min) with primary endothelial HUVECs on a 48-well plate seeded with 50 000 cells/well on an extracellular membrane mimicking MatrigelTM Matrix dissolved in DMEM. Study groups treated with fetal bovine serum and antibiotics were 5 and 10 µM KYP-2047 (+4 µM Tȕ4), 1:20 (0.625 µM) human recombinant POP (+4 µM Tȕ4)4 µM Tȕ4 (pos. control) and DMEM (neg. control). The wells were cultured and capillary formation photographed with a light microscope using a digital camera. All experiments were repeated four times, and each study group in wells was measured in triplicate. Enclosed capillaries were counted manually and statistical tests were performed. 7ȕ4 along with POP participated in the formation of AC-SDKP in the kidney homogenates. Cultures of primary endothelial cells on Matrigel resulted in clear capillary formation in Tȕ4 and POP groups. KYP-2047 had a strong POP-inhibitory activity on antiangiogenesis throughout the study resulting. Obviously, underlying mechanisms of angiogenesis and the function of the interaction between POP, Tȕ4 and Ac-SDKP in capillary formation require further studies.
  • Chen, Shuo (Helsingfors universitet, 2016)
    Tumor cells exhibit uncontrolled proliferation, which is supported and accelerated by a constant supply of nutrients carried by blood vessels. Tumor angiogenesis, the formation of new blood vessels, besides its contribution to tumor growth, also allows the dissemination of tumor cells into distant organs. In addition to the hematogenous routes, the tumor cells metastasize through lymphatic vasculature as well. Tumor-associated lymphangiogenesis, the formation of new lymphatic vessels, is a key process in this regard. Multiple growth factor pathways regulate angiogenesis and lymphangiogenesis. Among the most important vascular growth factors implicated in this regulation are vascular endothelial growth factors (VEGFs) and angiopoietin growth factors (Angs). It has been shown that targeting VEGF/VEGFR-2 pathway could inhibit tumor growth. Many studies during the last decade have demonstrated that attenuating VEGFR-3 function inhibits primary tumor growth and also metastasis. Selective antibodies against Ang2 were shown to inhibit tumor growth and metastasis in different tumor models in mice. However, the question regarding whether combining different therapeutic methods, namely anti-VEGFR-2, anti-VEGFR-3 and anti-Ang2, will have additive benefits in comparison to single-agent therapies still remains. We aimed to test the inhibitory effects of simultaneous targeting of all VEGF pathways and Ang2 on primary tumor growth in human lung carcinoma xenografts in immunodeficient mice. To achieve this, we used soluble VEGF – trap (Aflibercept), VEGF-C/D – trap and antibodies against Ang2. Our results show that triple therapy significantly improves the inhibition of primary tumor growth in comparison to monotherapies and dual therapies. Combination of all 3 treatments also improved the reduction of intra-tumor blood vessels and lymphatic vessels. The effects of triple targeting on controlling metastasis, however, was not significant in orthotopic breast cancer model, mainly due to great variation in tumor growth in this model. However, a clear trend of reduced metastasis in several organs (liver, kidneys) was observed. Overall, this study suggests that attenuating all VEGF pathways and Ang2 could improve the inhibitory effects of anti-angiogenic treatments.
  • Saba, Nabil F.; Vijayvargiya, Pooja; Vermorken, Jan B.; Rodrigo, Juan P.; Willems, Stefan M.; Zidar, Nina; de Bree, Remco; Mäkitie, Antti; Wolf, Greg T.; Argiris, Athanassios; Teng, Yong; Ferlito, Alfio (2022)
    Simple Summary Therapies for squamous cell carcinomas of the head and neck (SCCHN) have been rapidly evolving, initially with the inclusion of immunotherapy, but more recently with the consideration of anti-angiogenic therapies. Recent preclinical and clinical data reveal a strong correlation between vascular endothelial growth factor (VEGF) and the progression of SCCHN, with nearly 90% of these malignancies expressing VEGF. Our review article not only elaborates on the utility of anti-VEGF therapies on SCCHN but also its interaction with the immune environment. Furthermore, we detailed the current data on immunotherapies targeting SCCHN and how this could be coupled with anti-angiogenics therapies. Despite the lack of approved anti-angiogenic therapies in squamous cell carcinoma of the head and neck (SCCHN), preclinical and more recent clinical evidence support the role of targeting the vascular endothelial growth factor (VEGF) in this disease. Targeting VEGF has gained even greater interest following the recent evidence supporting the role of immunotherapy in the management of advanced SCCHN. Preclinical evidence strongly suggests that VEGF plays a role in promoting the growth and progression of SCCHN, and clinical evidence exists as to the value of combining this strategy with immunotherapeutic agents. Close to 90% of SCCHNs express VEGF, which has been correlated with a worse clinical prognosis and an increased resistance to chemotherapeutic agents. As immunotherapy is currently at the forefront of the management of advanced SCCHN, revisiting the rationale for targeting angiogenesis in this disease has become an even more attractive proposition.
  • Laitinen, Anita; Lampinen, Milla; Liedtke, Stefanie; Kilpinen, Lotta; Kerkela, Erja; Sarkanen, Jertta-Riina; Heinonen, Tuula; Kogler, Gesine; Laitinen, Saara (2016)
    Background aims. Cord blood (CB) is an attractive source of mesenchymal stromal cells (MSCs) because of its abundant availability and ease of collection. However, the success rate of generating CB-MSCs is low. In this study, our aim was to demonstrate the efficiency of our previously described method to obtain MSCs from CB and further characterize them and to study the effects of different culture conditions on MSCs. Methods. CB-MSC cultures were established in low oxygen (3%) conditions on fibronectin in 10% fetal bovine serum containing culture medium supplemented with combinations of growth factors. Cells were characterized for their adipogenic, osteogenic and chondrogenic differentiation capacity; phenotype; and HOX gene expression profile. The functionality of the cells cultured in different media was tested in vitro with angiogenesis and T-cell proliferation assays. Results. We demonstrate 87% efficacy in generating MSCs from CB. The established cells had typical MSC characteristics with reduced adipogenic differentiation potential and a unique HOX gene fingerprint. Growth factor rich medium and a 3% oxygen condition enhanced cell proliferation; however, the growth factor rich medium had a negative effect on the expression of CD90. Dexamethasone-containing medium improved the capacity of the cells to suppress T-cell proliferation, whereas the cells grown without dexamethasone were more able to support angiogenesis. Conclusions. Our results demonstrate that the composition of expansion medium is critical for the functionality of MSCs and should always be appropriately defined for each purpose.
  • Asghar, Muhammad Yasir; Törnquist, Kid (2020)
    Calcium (Ca2+) is perhaps the most versatile signaling molecule in cells. Ca2+ regulates a large number of key events in cells, ranging from gene transcription, motility, and contraction, to energy production and channel gating. To accomplish all these different functions, a multitude of channels, pumps, and transporters are necessary. A group of channels participating in these processes is the transient receptor potential (TRP) family of cation channels. These channels are divided into 29 subfamilies, and are differentially expressed in man, rodents, worms, and flies. One of these subfamilies is the transient receptor potential canonical (TRPC) family of channels. This ion channel family comprises of seven isoforms, labeled TRPC1-7. In man, six functional forms are expressed (TRPC1, TRPC3-7), whereas TRPC2 is a pseudogene; thus, not functionally expressed. In this review, we will describe the importance of the TRPC channels and their interacting molecular partners in the etiology of cancer, particularly in regard to regulating migration and invasion.