Exercise-induced expression of angiogenic growth factors in skeletal muscle and in capillaries of healthy and diabetic mice

Background. Diabetes has negative, and exercise training positive, effects on the skeletal muscle vasculature, but the mechanisms are not yet fully understood. In the present experiment the effects of running exercise on the mRNA expression of pro- and antiangiogenic factors were studied in healthy and diabetic skeletal muscle. The responses in capillaries and muscle fibers, collected from the muscle with laser capture microdissection, were also studied separately. Methods. Healthy and streptozotocin-induced diabetic mice were divided into sedentary and exercise groups. Exercise was a single bout of 1 h running on a treadmill. Gastrocnemius muscles were harvested 3 h and 6 h post exercise, and angiogenesis-related gene expressions were analyzed with real-time PCR. In addition to muscle homogenates, capillaries and muscle fibers were collected from the muscle with laser capture microdissection method and analyzed for vascular endothelial growth factor-A (VEGF-A) and thrombospondin-1 (TSP-1) mRNA expression. Results. Of the proangiogenic factors, VEGF-A and VEGF receptor-2 (VEGFR-2) mRNA expression increased significantly (P < 0.05) in healthy skeletal muscle 6 h post exercise. VEGF-B also showed a similar trend (P = 0.08). No significant change was observed post exercise in diabetic muscles in the expression of VEGF-A, VEGFR-2 or VEGF-B. The expression of angiogenesis inhibitor TSP-1 and angiogenic extracellular matrix protein Cyr61 were significantly increased in diabetic muscles (P < 0.05–0.01). Capillary mRNA expression resembled that in the muscle homogenates, however, the responses were greater in capillaries compared to muscle homogenates and pure muscle fibers. Conclusion. The present study is the first to report the effects of a single bout of exercise on the expression of pro- and antiangiogenic factors in diabetic skeletal muscle, and it provides novel data about the separate responses in capillaries and muscle fibers to exercise and diabetes. Diabetic mice seem to have lower angiogenic responses to exercise compared to healthy mice, and they show markedly increased expression of angiogenesis inhibitor TSP-1. Furthermore, exercise-induced VEGF-A expression was shown to be greater in capillaries than in muscle fibers.peerReviewe

HiPS-Endothelial Cells Acquire Cardiac Endothelial Phenotype in Co-culture With hiPS-Cardiomyocytes

Cell-cell interactions are crucial for organ development and function. In the heart, endothelial cells engage in bidirectional communication with cardiomyocytes regulating cardiac development and growth. We aimed to elucidate the organotypic development of cardiac endothelial cells and cardiomyocyte and endothelial cell crosstalk using human induced pluripotent stem cells (hiPSC). Single-cell RNA sequencing was performed with hiPSC-derived cardiomyocytes (hiPS-CMs) and endothelial cells (hiPS-ECs) in mono- and co-culture. The presence of hiPS-CMs led to increased expression of transcripts related to vascular development and maturation, cardiac development, as well as cardiac endothelial cell and endocardium-specific genes in hiPS-ECs. Interestingly, co-culture induced the expression of cardiomyocyte myofibrillar genes and MYL7 and MYL4 protein expression was detected in hiPS-ECs. Major regulators of BMP- and Notch-signaling pathways were induced in both cell types in co-culture. These results reflect the findings from animal studies and extend them to human endothelial cells, demonstrating the importance of EC-CM interactions during development.Peer reviewe

PROX1 transcription factor controls rhabdomyosarcoma growth, stemness, myogenic properties and therapeutic targets

Funding Information: ACKNOWLEDGMENTS. We would like to thank Dr. Tuomas Tammela and Dr. Monika Ehnmann for providing RMS cell lines and Dr. Jenny Högström for discussions and comments during the project. Kirsi Mattinen, Jefim Brodkin, Maxime Laird, Manon Gruchet, Ilse Paetau, Tanja Laakkonen, and Tapio Tainola are acknowledged for their excellent technical help. We also thank the Laboratory Animal Center at the University of Helsinki for expert animal care, the Biomedicum Imaging Unit for microscope support, the Biomedicum Functional Genomics Unit for the RNAseq experiments and the FIMM Technology Centre High Throughput Biomedicine for the drug sensitivity and resistance testing. Our first findings on PROX1 involvement in RMS and analyses presented in this study were made in the Translational Cancer Biology Program, University of Helsinki and Wihuri Research Institute. The work was funded by the Cancer Foundation Finland sr., Barncancerfonden, the Academy of Finland (grants 297245, 320185, 292816, 273817, and 307366), the Sigrid Jusélius Foundation, Children’s Cancer Foundation Väre, the Doctoral School of Biomedicine, iCAN Digital Precision Cancer Medicine Flagship, K. Albin Johanssons stiftelse sr., and The Hospital District of Helsinki, Uusimaa Research Grants (THY2019202 and TYH202102). Funding Information: We would like to thank Dr. Tuomas Tammela and Dr. Monika Ehnmann for providing RMS cell lines and Dr. Jenny Högström for discussions and comments during the project. Kirsi Mattinen, Jefim Brodkin, Maxime Laird, Manon Gruchet, Ilse Paetau, Tanja Laakkonen, and Tapio Tainola are acknowledged for their excellent technical help. We also thank the Laboratory Animal Center at the University of Helsinki for expert animal care, the Biomedicum Imaging Unit for microscope support, the Biomedicum Functional Genomics Unit for the RNAseq experiments and the FIMM Technology Centre High Throughput Biomedicine for the drug sensitivity and resistance testing. Our first findings on PROX1 involvement in RMS and analyses presented in this study were made in the Translational Cancer Biology Program, University of Helsinki and Wihuri Research Institute. The work was funded by the Cancer Foundation Finland sr., Barncancerfonden, the Academy of Finland (grants 297245, 320185, 292816, 273817, and 307366), the Sigrid Jusélius Foundation, Children’s Cancer Foundation Väre, the Doctoral School of Biomedicine, iCAN Digital Precision Cancer Medicine Flagship, K. Albin Johanssons stiftelse sr., and The Hospital District of Helsinki, Uusimaa Research Grants (THY2019202 and TYH202102). Publisher Copyright: Copyright © 2022 the Author(s).Rhabdomyosarcoma (RMS) is an aggressive pediatric soft-tissue cancer with features of skeletal muscle. Because of poor survival of RMS patients and severe long-term side effects of RMS therapies, alternative RMS therapies are urgently needed. Here we show that the prospero-related homeobox 1 (PROX1) transcription factor is highly expressed in RMS tumors regardless of their cell type of origin. We demonstrate that PROX1 is needed for RMS cell clonogenicity, growth and tumor formation. PROX1 gene silencing repressed several myogenic and tumorigenic transcripts and transformed the RD cell transcriptome to resemble that of benign mesenchymal stem cells. Importantly, we found that fibroblast growth factor receptors (FGFR) mediated the growth effects of PROX1 in RMS. Because of receptor cross-compensation, paralog-specific FGFR inhibition did not mimic the effects of PROX1 silencing, whereas a pan-FGFR inhibitor ablated RMS cell proliferation and induced apoptosis. Our findings uncover the critical role of PROX1 in RMS and offer insights into the mechanisms that regulate RMS development and growth. As FGFR inhibitors have already been tested in clinical phase I/II trials in other cancer types, our findings provide an alternative option for RMS treatment.Peer reviewe

Vascular Endothelial Growth Factor-B Induces a Distinct Electrophysiological Phenotype in Mouse Heart

Vascular endothelial growth factor B (VEGF-B) is a potentmediator of vascular, metabolic, growth, and stress responses in the heart, but the effects on cardiac muscle and cardiomyocyte function are not known. The purpose of this study was to assess the effects of VEGF-B on the energy metabolism, contractile, and electrophysiological properties of mouse cardiac muscle and cardiac muscle cells. In vivo and ex vivo analysis of cardiac-specific VEGF-B TG mice indicated that the contractile function of the TG hearts was normal. Neither the oxidative metabolism of isolated TG cardiomyocytes nor their energy substrate preference showed any difference to WT cardiomyocytes. Similarly, myocyte Ca2+ signaling showed only minor changes compared to WT myocytes. However, VEGF-B overexpression induced a distinct electrophysiological phenotype characterized by ECG changes such as an increase in QRSp time and decreases in S and R amplitudes. At the level of isolated TG cardiomyocytes, these changes were accompanied with decreased action potential upstroke velocity and increased duration (APD60-70). These changes were partly caused by downregulation of sodium current (INa) due to reduced expression of Nav1.5. Furthermore, TG myocytes had alterations in voltage-gated K + currents, namely decreased density of transient outward current (Ito) and total K + current (Ipeak). At the level of transcription, these were accompanied by downregulation of Kv channel-interacting protein 2 (Kcnip2), a knownmodulatory subunit for Kv4.2/3 channel. Cardiac VEGF-B overexpression induces a distinct electrophysiological phenotype including remodeling of cardiomyocyte ion currents, which in turn induce changes in action potential waveform and ECG.Peer reviewe

ORP2, a cholesterol transporter, regulates angiogenic signaling in endothelial cells

https://doi.org/10.1096/fj.202000202ROxysterol-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.Peer reviewe

Automated GMP production and long-term experience in radiosynthesis of CB(1)tracer [F-18]FMPEP-d(2)

Here, we describe the development of an in-house-built device for the fully automated multistep synthesis of the cannabinoid CB(1)receptor imaging tracer (3R,5R)-5-(3-([F-18]fluoromethoxy-d(2))phenyl)-3-(((R)-1-phenylethyl)amino)-1-(4-(trifluoromethyl)phenyl)pyrrolidin-2-one ([F-18]FMPEP-d(2)), following good manufacturing practices. The device is interfaced to a HPLC and a sterile filtration unit in a clean room hot cell. The synthesis involves the nucleophilic(18)F-fluorination of an alkylating agent and its GC purification, the subsequent(18)F-fluoroalkylation of a precursor molecule, the semipreparative HPLC purification of the(18)F-fluoroalkylated product, and its formulation for injection. We have optimized the duration and temperature of the(18)F-fluoroalkylation reaction and addressed the radiochemical stability of the formulated product. During the past 5 years (2013-2018), we have performed a total of 149 syntheses for clinical use with a 90% success rate. The activity yield of the formulated product has been 1.0 +/- 0.4 GBq starting from 11 +/- 2 GBq and the molar activity 600 +/- 300 GBq/mu mol at the end of synthesis

Exercise training improves adipose tissue metabolism and vasculature regardless of baseline glucose tolerance and sex

Introduction We investigated the effects of a supervised progressive sprint interval training (SIT) and moderate-intensity continuous training (MICT) on adipocyte morphology and adipose tissue metabolism and function; we also tested whether the responses were similar regardless of baseline glucose tolerance and sex. Research design and methods 26 insulin-resistant (IR) and 28 healthy participants were randomized into 2-week-long SIT (4-6x30 s at maximum effort) and MICT (40-60 min at 60% of maximal aerobic capacity (VO2peak)). Insulin-stimulated glucose uptake and fasting-free fatty acid uptake in visceral adipose tissue (VAT), abdominal and femoral subcutaneous adipose tissues (SATs) were quantified with positron emission tomography. Abdominal SAT biopsies were collected to determine adipocyte morphology, gene expression markers of lipolysis, glucose and lipid metabolism and inflammation. Results Training increased glucose uptake in VAT (pPeer reviewe

VEGF-B Promotes Endocardium-Derived Coronary Vessel Development and Cardiac Regeneration

cited By 1Background: Recent discoveries have indicated that, in the developing heart, sinus venosus and endocardium provide major sources of endothelium for coronary vessel growth that supports the expanding myocardium. Here we set out to study the origin of the coronary vessels that develop in response to vascular endothelial growth factor B (VEGF-B) in the heart and the effect of VEGF-B on recovery from myocardial infarction. Methods: We used mice and rats expressing a VEGF-B transgene, VEGF-B-gene-deleted mice and rats, apelin-CreERT, and natriuretic peptide receptor 3-CreERT recombinase-mediated genetic cell lineage tracing and viral vector-mediated VEGF-B gene transfer in adult mice. Left anterior descending coronary vessel ligation was performed, and 5-ethynyl-2'-deoxyuridine-mediated proliferating cell cycle labeling; flow cytometry; histological, immunohistochemical, and biochemical methods; single-cell RNA sequencing and subsequent bioinformatic analysis; microcomputed tomography; and fluorescent- and tracer-mediated vascular perfusion imaging analyses were used to study the development and function of the VEGF-B-induced vessels in the heart. Results: We show that cardiomyocyte overexpression of VEGF-B in mice and rats during development promotes the growth of novel vessels that originate directly from the cardiac ventricles and maintain connection with the coronary vessels in subendocardial myocardium. In adult mice, endothelial proliferation induced by VEGF-B gene transfer was located predominantly in the subendocardial coronary vessels. Furthermore, VEGF-B gene transduction before or concomitantly with ligation of the left anterior descending coronary artery promoted endocardium-derived vessel development into the myocardium and improved cardiac tissue remodeling and cardiac function. Conclusions: The myocardial VEGF-B transgene promotes the formation of endocardium-derived coronary vessels during development, endothelial proliferation in subendocardial myocardium in adult mice, and structural and functional rescue of cardiac tissue after myocardial infarction. VEGF-B could provide a new therapeutic strategy for cardiac neovascularization after coronary occlusion to rescue the most vulnerable myocardial tissue.Peer reviewe

Endothelial Cells Regulate Physiological Cardiomyocyte Growth via VEGFR2-Mediated Paracrine Signaling

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.Peer reviewe

VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart

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