Browsing by Subject "ANGIOGENESIS"

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  • Gousopoulos, Epameinondas; Proulx, Steven T.; Bachmann, Samia B.; Dieterich, Lothar C.; Scholl, Jeannette; Karaman, Sinem; Bianchi, Roberta; Detmar, Michael (2017)
    Secondary lymphedema is a common complication after cancer treatment, but the pathomechanisms underlying the disease remain unclear. Using a mouse tail lymphedema model, we found an increase in local and systemic levels of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C and identified CD68(+) macrophages as a cellular source. Surprisingly, overexpression of VEGF-C in a transgenic mouse model led to aggravation of lymphedema with increased immune cell infiltration and vascular leakage compared with wild-type littermates. Conversely, blockage of VEGF-C by overexpression of soluble VEGF receptor-3 reduced edema development, diminishing inflammation and blood vascular leakage. Similar findings were obtained in a hind limb lymph node excision lymphedema model. Flow cytometry analyses and immunofluorescence stainings in lymphedematic tissue showed that VEGF receptor-3 expression was restricted to lymphatic endothelial cells. Our data suggest that endogenous VEGF-C causes blood vascular leakage and fluid influx into the tissue, thus actively contributing to edema formation. These data may provide the basis for future clinical therapeutic approaches.
  • Chu, Man; Li, Taotao; Shen, Bin; Cao, Xudong; Zhong, Haoyu; Zhang, Luqing; Zhou, Fei; Ma, Wenjuan; Jiang, Haijuan; Xie, Pancheng; Liu, Zhengzheng; Dong, Ningzheng; Xu, Ying; Zhao, Yun; Xu, Guoqiang; Lu, Peirong; Luo, Jincai; Wu, Qingyu; Alitalo, Kari; Koh, Gou Young; Adams, Ralf H.; He, Yulong (2016)
    Mechanisms underlying the vein development remain largely unknown. Tie2 signaling mediates endothelial cell (EC) survival and vascular maturation and its activating mutations are linked to venous malformations. Here we show that vein formation are disrupted in mouse skin and mesentery when Tie2 signals are diminished by targeted deletion of Tek either ubiquitously or specifically in embryonic ECs. Postnatal Tie2 attenuation resulted in the degeneration of newly formed veins followed by the formation of haemangioma-like vascular tufts in retina and venous tortuosity. Mechanistically, Tie2 insufficiency compromised venous EC identity, as indicated by a significant decrease of COUP-TFII protein level, a key regulator in venogenesis. Consistently, angiopoietin-1 stimulation increased COUP-TFII in cultured ECs, while Tie2 knockdown or blockade of Tie2 downstream PI3K/Akt pathway reduced COUP-TFII which could be reverted by the proteasome inhibition. Together, our results imply that Tie2 is essential for venous specification and maintenance via Akt mediated stabilization of COUP-TFII.
  • Rautiola, Juhana; Lampinen, Anita; Mirtti, Tuomas; Ristimaki, Ari; Joensuu, Heikki; Bono, Petri; Saharinen, Pipsa (2016)
    The Angiopoietin-2 (Ang2, Angpt2) growth factor is a context-dependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase and known to promote tumour angiogenesis and metastasis. Angiopoietin antagonists have been tested in clinical cancer trials in combination with VEGF-based anti-angiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. Here, we evaluated, using immunohistochemistry, the expression of Ang2, CD31 and the cell proliferation marker Ki-67 in the primary kidney cancer from 136 mRCC patients, who received first-line sunitinib after nephrectomy. Ang2 protein expression was restrained to RCC tumour vessels, and correlated with tumour vascularization and response to sunitinib. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS, 15.7 vs. 28.5 months, P = 0.015). In summary, in this study to investigate endothelial Ang2 in mRCC patients treated with first-line sunitinib, high cancer Ang2 expression was associated with the CBR, but not PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS.
  • Martinez-Corral, Ines; Zhang, Yan; Petkova, Milena; Ortsater, Henrik; Sjoberg, Sofie; Castillo, Sandra D.; Brouillard, Pascal; Libbrecht, Louis; Saur, Dieter; Graupera, Mariona; Alitalo, Kari; Boon, Laurence; Vikkula, Miikka; Mäkinen, Taija (2020)
    Lymphatic malformations (LMs) are debilitating vascular anomalies presenting with large cysts (macrocystic) or lesions that infiltrate tissues (microcystic). Cellular mechanisms underlying LM pathology are poorly understood. Here we show that the somatic PIK3CA(H1047R) mutation, resulting in constitutive activation of the p110 alpha PI3K, underlies both macrocystic and microcystic LMs in human. Using a mouse model of PIK3CA(H1047R)-driven LM, we demonstrate that both types of malformations arise due to lymphatic endothelial cell (LEC)-autonomous defects, with the developmental timing of p110 alpha activation determining the LM subtype. In the postnatal vasculature, PIK3CA(H1047R) promotes LEC migration and lymphatic hypersprouting, leading to microcystic LMs that grow progressively in a vascular endothelial growth factor C (VEGF-C)-dependent manner. Combined inhibition of VEGF-C and the PI3K downstream target mTOR using Rapamycin, but neither treatment alone, promotes regression of lesions. The best therapeutic outcome for LM is thus achieved by co-inhibition of the upstream VEGF-C/VEGFR3 and the downstream PI3K/mTOR pathways. Lymphatic malformation (LM) is a debilitating often incurable vascular disease. Using a mouse model of LM driven by a disease-causative PIK3CA mutation, the authors show that vascular growth is dependent on the upstream lymphangiogenic VEGF-C signalling, permitting effective therapeutic intervention.
  • Kallio, Pauliina; Jokinen, Elina; Högström, Jenny; Das, Suvendu; Heino, Sarika; Lähde, Marianne; Brodkin, Jefim; Korhonen, Emilia A.; Alitalo, Kari (2020)
    Abnormal vasculature in tumors leads to poor tissue perfusion and cytostatic drug delivery. Although drugs inducing vascular normalization, for example, angiopoietin-2 (Ang2)-blocking antibodies, have shown promising results in preclinical tumor models, clinical studies have so far shown only little efficacy. Because Ang2 is known to play a protective role in stressed endothelial cells, we tested here whether Ang2 blocking could enhance radiation-induced tumor vascular damage. Tumor-bearing mice were treated with anti-Ang2 antibodies every 3 or 4 days starting 3 days before 3 x 2 Gy or 4 x 0.5 Gy whole-body or tumor-focused radiation. Combination treatment with anti-Ang2 and radiation improved tumor growth inhibition and extended the survival of mice with melanoma or colorectal tumors. Single-cell RNA-sequencing revealed that Ang2 blocking rescued radiation-induced decreases inT cells and cells of the monocyte/macrophage lineage. In addition, anti-Ang2 enhanced radiation-induced apoptosis in cultured endothelial cells. In vivo, combination treatment decreased tumor vasculature and increased tumor necrosis in comparison with tumors treated with monotherapies. These results suggest that a combination of Ang2-blocking antibodies with 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 radiation in combination with Ang2-blocking antibodies to improve the overall outcome of cancer treatment.
  • Hemanthakumar, Karthik Amudhala; Fang, Shentong; Anisimov, Andrey; Mäyränpää, Mikko I.; Mervaala, Eero; Kivelä, Riikka (2021)
    Aging, obesity, hypertension, and physical inactivity are major risk factors for endothelial dysfunction and cardiovascular disease (CVD). We applied fluorescence-activated cell sorting (FACS), RNA sequencing, and bioinformatic methods to investigate the common effects of CVD risk factors in mouse cardiac endothelial cells (ECs). Aging, obesity, and pressure overload all upregulated pathways related to TGF-beta signaling and mesenchymal gene expression, inflammation, vascular permeability, oxidative stress, collagen synthesis, and cellular senescence, whereas exercise training attenuated most of the same pathways. We identified collagen chaperone Serpinhl (also called as Hsp47) to be significantly increased by aging and obesity and repressed by exercise training. Mechanistic studies demonstrated that increased SERPINH1 in human ECs induced mesenchymal properties, while its silencing inhibited collagen deposition. Our data demonstrate that CVD risk factors significantly remodel the transcriptomic landscape of cardiac ECs inducing inflammatory, senescence, and mesenchymal features. SERPINH1 was identified as a potential therapeutic target in ECs.
  • Leppänen, Veli-Matti; Brouillard, Pascal; Korhonen, Emilia A.; Sipilä, Tuomas; Jha, Sawan Kumar; Revencu, Nicole; Labarque, Veerle; Fastré, Elodie; Schlögel, Matthieu; Ravoet, Marie; Singer, Amihood; Luzzatto, Claudia; Angelone, Donatella; Crichiutti, Giovanni; D’Elia, Angela; Kuurne, Jaakko; Elamaa, Harri; Koh, Gou Young; Saharinen, Pipsa; Vikkula, Miikka; Alitalo, Kari (2020)
    Lymphedema can occur when tissue fluid cannot enter or leaks from the lymphatic system into surrounding tissues. Some genetic causes of primary lymphedema are known, but these currently explain a minority of cases. Previous studies have shown that dominant-negative mutations in angiopoietin 2 (ANGPT2), which is involved in lymphatic vessel formation and maturation, promote lymphangiogenesis in mice. Leppänen et al. now show that inactivating mutations in angiopoietin 2 associate with primary lymphedema in humans.Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain <30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor–like domains 1 and 2) receptor complex and α5β1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)–ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin α5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.
  • Liu, Zehua; Li, Yunzhan; Li, Wei; Lian, Wenhua; Kemell, Marianna; Hietala, Sami; Figueiredo, Patricia; Li, Li; Mäkilä, Ermei; Ma, Ming; Salonen, Jarno; Hirvonen, Jouni T.; Liu, Dongfei; Zhang, Hongbo; Deng, Xianming; Santos, Helder A. (2019)
    Here, an oxidation/acid dual-responsive nanohybrids/ark system was produced. The microfluidics-produced nanohybrids endow the system with an orchestrated cascade from wound detection, reactive oxygen species scavenging, drug release to hydrogel formation. The drug release behavior imitates the dynamic wound healing process, thus rendering an enhanced bio-mimetic regeneration.
  • Falke, J.; Parkkinen, J.; Vaahtera, L.; Hulsbergen-van de Kaa, C. A.; Oosterwijk, E.; Witjes, J. A. (2018)
    Objective. To evaluate the antitumor effect of cyclodextrin-curcumin complex (CDC) on human and rat urothelial carcinoma cells in vitro and to evaluate the effect of intravesical instillations of CDC, BCG, and the combination in vivo in the AY-F344 orthotopic bladder cancer rat model. Curcumin has anticarcinogenic activity on urothelial carcinoma and is therefore under investigation for the treatment of non-muscle invasive bladder cancer. Curcumin and BCG share immunomodulating pathways against urothelial carcinoma. Methods. Curcumin was complexed with cyclodextrin to improve solubility. Four human urothelial carcinoma cell lines and the AY-27 rat cell line were exposed to various concentrations of CDC in vitro. For the in vivo experiment, the AY-27 orthotopic bladder cancer F344 rat model was used. Rats were treated with consecutive intravesical instillations of CDC, BCG, the combination of CDC+BCG, or NaCl as control. Results. CDC showed a dose-dependent antiproliferative effect on all human urothelial carcinoma cell lines tested and the rat AY-27 urothelial carcinoma cell line. Moreover, intravesical treatment with CDC and CDC+BCG results in a lower percentage of tumors (60% and 68%, respectively) compared to BCG (75%) or control (85%). This difference with placebo was not statistically significant (p=0.078 and 0.199, respectively). However, tumors present in the placebo and BCG-treated rats were generally of higher stage. Conclusions. Cyclodextrin-curcumin complex showed an antiproliferative effect on human and rat urothelial carcinoma cell lines in vitro. In the aggressive orthotopic bladder cancer rat model, we observed a promising effect of CDC treatment and CDC in combination with BCG.
  • Aalto-Setälä, Laura; Uppstu, Peter; Sinitsyna, Polina; Lindfors, Nina C.; Hupa, Leena (2021)
    The silicate-based bioactive glass S53P4 is clinically used in bone regenerative applications in granule form. However, utilization of the glass in scaffold form has been limited by the high tendency of the glass to crystallize during sintering. Here, careful optimization of sintering parameters enabled the manufacture of porous amorphous S53P4 scaffolds with a strength high enough for surgical procedures in bone applications (5 MPa). Sintering was conducted in a laboratory furnace for times ranging from 25 to 300 min at 630 degrees C, i.e., narrowly below the commencement of the crystallization. The phase composition of the scaffolds was verified with XRD, and the ion release was tested in vitro and compared with granules in continuous flow of Tris buffer and simulated body fluid (SBF). The amorphous, porous S53P4 scaffolds present the possibility of using the glass composition in a wider range of applications.
  • Al-Samadi, Ahmed; Moossavi, Shirin; Salem, Abdelhakim; Sotoudeh, Masoud; Tuovinen, Sarianna M.; Konttinen, Yrjo T.; Salo, Tuula; Bishehsari, Faraz (2016)
    Colorectal cancer (CRC) is one of the most common cancers in both genders. Even though interleukin (IL)-17A was shown to play an important role in intestinal tumourigenesis and CRC, other IL-17 family members were not studied well. We therefore studied the expression of IL-17 cytokine family members in CRC. Ten healthy colons and ten CRC mucosa were immunostained for IL-17B, IL-17C, IL-17E, and IL-17F, and their receptors IL-17RA, IL-17RB, and IL-17RC. Double immunofluorescence staining of the CRC mucosa was done for IL-17B with markers of neutrophils, endothelial cells, macrophages, T cells, mast cells, or fibroblasts. While IL-17B was increased in CRC with a strong presence both in the epithelial and stromal compartments, IL-17C showed different expression depending on the grade of differentiation and IL-17E remained unchanged. In contrast, IL-17F was decreased in CRC compared to healthy control. Colon epithelial cells stained positive for IL-17RA, IL-17RB, and IL-17RC in both healthy control and CRC. Neutrophils were the main source of IL-17B in the stroma. IL-17 family members demonstrated distinct expression patterns in CRC, suggesting a differential role exerted by each member in colon carcinogenesis.
  • Auvinen, Vili-Veli; Merivaara, Arto; Kiiskinen, Jasmi; Paukkonen, Heli; Laurén, Patrick; Hakkarainen, Tiina; Koivuniemi, Raili; Sarkanen, Riina; Ylikomi, Timo; Laaksonen, Timo; Yliperttula, Marjo (2019)
    The aim of this study was to evaluate the effects of two nanofibrillated cellulose (NFC) hydrogels on two human derivatives during freeze-drying. Native NFC hydrogel is a suitable platform to culture 3D cell spheroids and a hydrogel processed further, called anionic NFC (ANFC) hydrogel, is an excellent platform for controlled release of proteins. Moreover, it has been shown to be compatible with freeze-drying when correct lyoprotectants are implemented. Freeze-drying is a method, where substance is first frozen, and then vacuum dried trough sublimation of water in order to achieve dry matter without the loss of the original three-dimensional structures. The first chosen human derivative was adipose tissue extract (ATE) which is a cell-free growth factor-rich preparation capable of promoting growth of regenerative cells. The release of growth factors from the freeze-dried mixture of ATE and ANFC was compared to that of non-freeze-dried control mixtures. The release profiles remained at the same level after freeze-drying. The second derivative was hepatocellular carcinoma (HepG2) cell spheroids which were evaluated before and after freeze-drying. The 3D structure of the HepG2 cell spheroids was preserved and the spheroids retained 18% of their metabolic activity after rehydration. However, the freeze-dried and rehydrated HepG2 cell spheroids did not proliferate and the cell membrane was damaged by fusion and formation of crystals.
  • 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.
  • Hakanpaa, Laura; Sipila, Tuomas; Leppanen, Veli-Matti; Gautam, Prson; Nurmi, Harri; Jacquemet, Guillaume; Eklund, Lauri; Ivaska, Johanna; Alitalo, Kari; Saharinen, Pipsa (2015)
  • Blasiak, Janusz; Watala, Cezary; Tuuminen, Raimo; Kivinen, Niko; Koskela, Ali; Uusitalo-Jarvinen, Hannele; Tuulonen, Anja; Winiarczyk, Mateusz; Mackiewicz, Jerzy; Zmorzynski, Szymon; Filip, Agata; Kaarniranta, Kai (2019)
    MicroRNAs (miRNAs) regulate gene expression; many of them act in the retinal pigment epithelium (RPE), and RPE degeneration is known to be a critical factor in age-related macular degeneration (AMD). Repeated injections with anti-VEGFA (vascular endothelial growth factor A) are the only effective therapy in wet AMD. We investigated the correlation between the expression of 18 miRNAs involved in the regulation of the VEGFA gene in serum of 76 wet AMD patients and 70 controls. Efficacy of anti-VEGFA treatment was evaluated by counting the number of injections delivered up to 12 years. In addition, we compared the relative numbers of deaths in patient with AMD and control groups. We observed a decreased expression of miR-34-5p, miR-126-3p, miR-145-5p and miR-205-5p in wet AMD patients as compared with controls. These miRNAs are involved in the regulation of angiogenesis, cytoprotection and protein clearance. No miRNA was significantly correlated with the treatment outcome. Wet AMD patients had greater mortality than controls, and their survival was inversely associated with the number of anti-VEGFA injections per year. No association was observed between miRNA expression and mortality. Our study emphasizes the need to clarify the role of miRNA regulation in AMD pathogenesis.
  • Helle, Emmi; Ampuja, Minna; Antola, Laura; Kivelä, Riikka (2020)
    The vascular system is essential for the development and function of all organs and tissues in our body. The molecular signature and phenotype of endothelial cells (EC) are greatly affected by blood flow-induced shear stress, which is a vital component of vascular development and homeostasis. Recent advances in differentiation of ECs from human induced pluripotent stem cells (hiPSC) have enabled development of in vitro experimental models of the vasculature containing cells from healthy individuals or from patients harboring genetic variants or diseases of interest. Here we have used hiPSC-derived ECs and bulk- and single-cell RNA sequencing to study the effect of flow on the transcriptomic landscape of hiPSC-ECs and their heterogeneity. We demonstrate that hiPS-ECs are plastic and they adapt to flow by expressing known flow-induced genes. Single-cell RNA sequencing showed that flow induced a more homogenous and homeostatically more stable EC population compared to static cultures, as genes related to cell polarization, barrier formation and glucose and fatty acid transport were induced. The hiPS-ECs increased both arterial and venous markers when exposed to flow. Interestingly, while in general there was a greater increase in the venous markers, one cluster with more arterial-like hiPS-ECs was detected. Single-cell RNA sequencing revealed that not all hiPS-ECs are similar even after sorting, but exposing them to flow increases their homogeneity. Since hiPS-ECs resemble immature ECs and demonstrate high plasticity in response to flow, they provide an excellent model to study vascular development.
  • Hjort, Line; Moller, Sofie Lykke; Minja, Daniel; Msemo, Omari; Nielsen, Birgitte Bruun; Christensen, Dirk Lund; Theander, Thor; Nielsen, Karsten; Larsen, Lise Grupe; Grunnet, Louise Groth; Groop, Leif; Prasad, Rashmi; Lusingu, John; Schmiegelow, Christentze; Bygbjerg, Ib C. (2019)
    Purpose Low-income and middle-income countries such as Tanzania experience a high prevalence of noncommunicable diseases (NCDs), including anaemia. Studying if and how anaemia affects growth, placenta development, epigenetic patterns and newborns' risk of NCDs may provide approaches to prevent NCDs. Participants The FOETALforNCD (FOetal Exposure and Epidemiological Transitions: the role of Anaemia in early Life for Non-Communicable Diseases in later life) Study is a population-based preconception, pregnancy and birth cohort study (n= 1415, n= 538, n= 427, respectively), conducted in a rural region of North-East Tanzania. All participants were recruited prior to conception or early in pregnancy and followed throughout pregnancy as well as at birth. Data collection included: maternal blood, screening for NCDs and malaria, ultrasound in each trimester, neonatal anthropometry at birth and at 1 month of age, cord blood, placental and cord biopsies for stereology and epigenetic analyses. Findings to date At preconception, the average age, body mass index and blood pressure of the women were 28 years, 23 kg/m(2) and 117/75 mm Hg, respectively. In total, 458 (36.7%) women had anaemia (haemoglobin Hb <12 g/dL) and 34 (3.6%) women were HIV-positive at preconception. During pregnancy 359 (66.7%) women had anaemia of which 85 (15.8%) women had moderate-tosevere anaemia (Hb = 9 g/dL) and 33 (6.1%) women had severe anaemia (Hb = 8 g/dL). In total, 185 (34.4%) women were diagnosed with malaria during pregnancy. Future plans The project will provide new knowledge on how health, even before conception, might modify the risk of developing NCDs and how to promote better health during pregnancy. The present project ended data collection 1 month after giving birth, but follow-up is continuing through regular monitoring of growth and development and health events according to the National Road Map Strategic Plan in Tanzania. This data will link fetal adverse event to childhood development, and depending on further grant allocation, through a life course follow-up.
  • Johns, Scott C.; Yin, Xin; Jeltsch, Michael; Bishop, Joseph R.; Schuksz, Manuela; El Ghazal, Roland; Wilcox-Adelman, Sarah A.; Alitalo, Kari; Fuster, Mark M. (2016)
    Rationale: Lymphatic vessel growth is mediated by major prolymphangiogenic factors, such as vascular endothelial growth factor (VEGF-C) and VEGF-D, among other endothelial effectors. Heparan sulfate is a linear polysaccharide expressed on proteoglycan core proteins on cell membranes and matrix, playing roles in angiogenesis, although little is known about any function(s) in lymphatic remodeling in vivo. Objective: To explore the genetic basis and mechanisms, whereby heparan sulfate proteoglycans mediate pathological lymphatic remodeling. Methods and Results: Lymphatic endothelial deficiency in the major heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in glycan-chain sulfation) was associated with reduced lymphangiogenesis in pathological models, including spontaneous neoplasia. Mouse mutants demonstrated tumor-associated lymphatic vessels with apoptotic nuclei. Mutant lymphatic endothelia demonstrated impaired mitogen (Erk) and survival (Akt) pathway signaling and reduced VEGF-C-mediated protection from starvation-induced apoptosis. Lymphatic endothelial-specific Ndst1 deficiency (in Ndst1(f/f)Prox1(+/CreERT2) mice) was sufficient to inhibit VEGF-C-dependent lymphangiogenesis. Lymphatic heparan sulfate deficiency reduced phosphorylation of the major lymphatic growth receptor VEGF receptor-3 in response to multiple VEGF-C species. Syndecan-4 was the dominantly expressed heparan sulfate proteoglycan in mouse lymphatic endothelia, and pathological lymphangiogenesis was impaired in Sdc4((-/-)) mice. On the lymphatic cell surface, VEGF-C induced robust association between syndecan-4 and VEGF receptor-3, which was sensitive to glycan disruption. Moreover, VEGF receptor-3 mitogen and survival signaling was reduced in the setting of Ndst1 or Sdc4 deficiency. Conclusions: These findings demonstrate the genetic importance of heparan sulfate and the major lymphatic proteoglycan syndecan-4 in pathological lymphatic remodeling. This may introduce novel future strategies to alter pathological lymphatic-vascular remodeling.
  • Crona, Daniel J.; Skol, Andrew D.; Leppänen, Veli-Matti; Glubb, Dylan M.; Etheridge, Amy S.; Hilliard, Eleanor; Pena, Carol E.; Peterson, Yuri K.; Klauber-DeMore, Nancy; Alitalo, Kari K.; Innocenti, Federico (2019)
    Molecular markers of sorafenib efficacy in patients with metastatic renal cell carcinoma (mRCC) are not available. The purpose of this study was to discover genetic markers of survival in patients with mRCC treated with sorafenib. Germline variants from 56 genes were genotyped in 295 patients with mRCC. Variant-overall survival (OS) associations were tested in multivariate regression models. Mechanistic studies were conducted to validate clinical associations. VEGFA rs1885657, ITGAV rs3816375, and WWOX rs8047917 (sorafenib arm), and FLT4 rs307826 and VEGFA rs3024987 (sorafenib and placebo arms combined) were associated with shorter OS. FLT4 rs307826 increased VEGFR-3 phosphorylation, membrane trafficking, and receptor activation. VEGFA rs1885657 and rs58159269 increased transcriptional activity of the constructs containing these variants in endothelial and RCC cell lines, and VEGFA rs58159269 increased endothelial cell proliferation and tube formation. FLT4 rs307826 and VEGFA rs58159269 led to reduced sorafenib cytotoxicity. Genetic variation in VEGFA and FLT4 could affect survival in sorafenib-treated patients with mRCC. These markers should be examined in additional malignancies treated with sorafenib and in other angiogenesis inhibitors used in mRCC. Significance: Clinical and mechanistic data identify germline genetic variants in VEGFA and FLT4 as markers of survival in patients with metastatic renal cell carcinoma.
  • Tiainen, Leena; Korhonen, Emilia A.; Leppänen, Veli-Matti; Luukkaala, Tiina; Hämäläinen, Mari; Tanner, Minna; Lahdenperä, Outi; Vihinen, Pia; Jukkola, Arja; Karihtala, Peeter; Aho, Sonja; Moilanen, Eeva; Alitalo, Kari; Kellokumpu-Lehtinen, Pirkko-Liisa (2019)
    BackgroundAngiopoietin growth factors (Angs) regulate angiogenesis and lymphangiogenesis by binding to the endothelial Tie2 receptor. Ang2 expression is elevated in tissue hypoxia and inflammation, which also induce cleavage of the extracellular domain of the orphan Tie1 receptor. Here we have examined if the concentrations of Ang2 and the soluble extracellular domain of Tie1 in patient plasma are associated with the prognosis of patients with metastatic breast cancer.MethodsPlasma Tie1 and Ang2 levels were measured in metastatic breast cancer patients treated in a phase II trial with a taxane-bevacizumab combination chemotherapy in the first-line treatment setting. They were analyzed before treatment, after 6weeks and 6months of treatment, and at the final study visit. Using the median concentrations as cutoffs, Tie1 and Ang2 data were dichotomized into low and high concentration groups. Additionally, we analyzed Tie1 concentrations in plasma from 10 healthy women participating in a breast cancer primary prevention study.ResultsPlasma samples were available from 58 (89%) of the 65 patients treated in the trial. The baseline Tie1 levels of the healthy controls were significantly lower than those of the metastatic patients (p