Cardiac endothelial cells : potential therapeutic targets in heart disease

Cardiovascular diseases (CVD) rank as a number one cause for mortality and accounts for one third of the deaths in several OECD countries (OECD, 2015). According to WHO, CVD pathology is characterised by impaired coronary vasculature associated with cardiac dysfunction, which often results in heart failure (Mendis S, 2011). Although the outlook for prevention and management of CVD risk factors is advancing, the extent of CVD mortality and morbidity remains relatively high (Mendis S, 2011) and the clinical prognosis of heart failure remains poorer than most of the cancers (Braunwald, 2015). Better understanding of the cellular and molecular links between the coronary vasculature and cardiac function under physiological and pathological conditions would enhance the development of personalized targeted therapies for CVD. In this thesis, my main objective is to define mechanistic insights how proangiogenic cues like VEGF-B or PlGF promote coronary angiogenesis -mediated physiological cardiac hypertrophy and to characterise the effect of CVD risk factors (aging, obesity, physical inactivity, and pressure overload) on cardiac endothelial cells (ECs) and cardiac function. We have applied molecular, biochemical, imaging and gene delivery methods to elucidate the phenotypes and molecular mechanisms in in vivo and in vitro model systems. In the study I, we showed that AAV9-VEGF-B overexpression or endothelial deletion of VEGFR1 increased the bioavailability of the endogenous VEGF to activate VEGFR2 in ECs promoting coronary angiogenesis. Importantly, this indirect activation of VEGFR2 is limited by endogenous levels of VEGF, and it did not promote vascular leakage. VEGFR2 activation induced expression of e.g., Dll4, Notch, Apln, Apj, Klk8 and Adam12, indicating activation of Notch and apelin signalling. Adam12 and Klk8, in turn, have been shown to induce shredding of Hb-egf and Nrg1 on the cell surface, leading to activation of ErbB receptors present in cardiomyocytes (CMCs). The findings of this study demonstrate a bidirectional crosstalk between ECs and CMCs via VEGFR2, NOTCH and ErbB signalling pathways. In the study II, overexpression of VEGF-B promoted cardiac EC activation throughout the heart, detected by lineage tracing using AplnCreERT2;Tdtomato reporter mice. However, the VEGF-B -induced EC proliferation was mainly concentrated to the subendocardial myocardium, which was detected by EdU labelling of proliferating cells and staining for a proliferation marker Ki67. In the Study II, my main contribution was the development and optimization of cardiac EC isolation for single-cell RNA sequencing. In this study, the main novel finding was that VEGF-B can promote coronary vessel formation from the endocardium during development and after myocardial infarction, which was accompanied by protection from the ischemic insult. In conclusion, the Studies I and II demonstrated the coronary angiogenesis -mediated physiological cardiomyocyte growth is mediated via VEGFR2-NOTCH-ErbB pathways and involves delicate bidirectional crosstalk between ECs and CMC. In the study III, the effects of CVD risk factors aging, obesity and pressure overload, and exercise training as a physiological stimulus, was studied on cardiac endothelial cells of C57Bl/6J mice. Pure and viable cardiac ECs were isolated and analysed by RNA sequencing and various bioinformatics tools. The data demonstrated that CVD risk factors significantly decreased the number of ECs in the heart as well as the coronary vascular density and cardiac function. Importantly, exercise training improved all of these parameters compared to the sedentary control mice. The next generation RNA sequencing revealed that CVD risk factors significantly remodelled the cardiac EC transcriptome and upregulated several genes and pathways related to inflammation, oxidative stress, TGF-b signalling, vascular permeability, endothelial to mesenchymal transition (EndMT) and cellular senescence, whereas exercise training inhibited most of the same pathways, demonstrating the beneficial role of exercise training on ECs and vasculature. Exercise training also promoted blood vessel development, vascular stability and homeostasis and cell-cell junctions. The gene overlap analysis of the differentially expressed genes in the different data sets revealed SerpinH1 as one of the commonly regulated gene. SerpinH1 was significantly induced by aging and obesity and repressed by exercise training. In vitro studies in human coronary arterial endothelial cells (HCAEC) showed that overexpression of SERPINH1 increased the cell size, induced the expression of EndMT and senescence related transcripts, repressed EC genes and enhanced migration in a wound healing assay. Silencing of SERPINH1 in HCAECs, in turn, completely blocked cell proliferation and decreased collagen deposition and wound healing. As SERPINH1 has previously been linked to fibrosis in other tissues and cell types, and here we found it in all ECs throughout the human and mouse heart, it might be a potential target for the treatment of CVD. The ECs are not often considered as therapeutic targets, even though in many cases heart problems arise secondary to vascular defects (Heusch et al., 2014). The results from this thesis suggest that cardiac ECs are highly adaptive to physiological stimuli and maladaptive to pathological stressors. These findings would help to develop innovative and new therapeutic opportunities to treat heart diseases.Sydän- ja verisuonitaudit (CVD) aiheuttavat kolmanneksen kuolemantapauksista useissa OECD-maissa. Vaikka CVD-riskitekijöiden ehkäisy ja hallinta on kehittynyt merkittävästi viime vuosina, CVD-kuolleisuuden ja -sairastavuuden laajuus on edelleen korkea, ja sydämen vajaatoiminnan kliininen ennuste on edelleen huonompi kuin useimpien syöpien. Sepelvaltimoiden verisuoniston seinämiä reunustavien endoteelisolujen ja sydämen toiminnan välisten solu- ja molekyyliyhteyksien parempi ymmärtäminen fysiologisissa ja patologisissa olosuhteissa parantaisi CVD:n hoitojen kehittämistä. Tässä väitöskirjassa päätavoitteeni oli tutkia miten proangiogeeniset kasvutekijät, kuten VEGF-B tai PlGF, edistävät sepelvaltimoiden angiogeneesi-välitteistä fysiologista sydämen hypertrofiaa sekä selvittää CVD-riskitekijöiden (ikääntyminen, liikalihavuus, fyysinen inaktiivisuus ja verenpainekuormitus) vaikutusta sydämen endoteelisoluihin ja sydämen toimintaan. Tutkimuksessani käytin molekyylibiologisia ja biokemiallisia analyysejä, erilaisia kuvantamismenetelmiä sekä muuntogeenisiä ja geeniterapia vektoreita fenotyyppien ja molekyylimekanismien selvittämiseksi in vivo eläinmalleissa sekä in vitro solumalleissa. Tutkimuksessa I osoitimme, että verisuonten endoteelisolujen ja sydänsolujen välillä tapahtuva kaksisuuntainen kommunikaatio VEGFR2-, NOTCH- ja ErbB-signalointireittien kautta säätelee verisuonten ja sydänsolujen kasvua suhteessa toisiinsa. Tämä auttaa ymmärtämään paremmin sydämen fysiologisen kasvun mekanismeja. Tutkimuksessa II osoitimme, että VEGF-B:n yliekspressio edistää sepelvaltimoiden verisuonten muodostumista endokardiumista kehityksen aikana ja sydäninfarktin jälkeen, mikä suojaa sydäntä hapenpuutteen aiheuttamalta vauriolta. Tutkimuksessa III tutkittiin CVD-riskitekijöiden ikääntymisen, liikalihavuuden ja sydämen painekuormituksen sekä fysiologisena ärsykkeenä toimivan liikuntaharjoittelun vaikutuksia hiirten sydämen endoteelisoluihin. Tulokset osoittivat, että CVD-riskitekijät vähensivät merkittävästi sydämen endoteelisolujen määrää sekä sepelvaltimoiden verisuonitiheyttä ja heikensivät sydämen toimintaa. Tärkeä havainto oli, että liikuntaharjoittelu paransi kaikkia näitä parametreja verrattuna inaktiivisiin kontrollihiiriin. RNA-sekvensointi osoitti, että CVD-riskitekijät muokkasivat merkittävästi sydämen endoteelisolujen geenien ilmenemistä ja lisäsivät useiden geenien ja signaalireittien ilmenemistä, jotka liittyvät mm. tulehdukseen, oksidatiiviseen stressiin, verisuonten läpäisevyyteen, endoteelin ja mesenkymaalisen solutyypin väliseen siirtymään (EndMT) ja solujen vanhenemiseen. Liikuntaharjoittelu puolestaan esti suurimman osan samoista reiteistä, mikä osoittaa liikuntaharjoittelun terveyttä edistävän roolin endoteelisolujen ja verisuoniston kannalta. Tulokset paljastivat SerpinH1 geenin lisääntyvän ikääntymisen ja ylipainon vaikutuksesta ja vähenevän liikuntaharjoittelulla. Koska SERPINH1 on aiemmin yhdistetty fibroosiin muissa kudoksissa ja solutyypeissä, se on mahdollisesti potentiaalinen lääkehoidon kohde sydän- ja verisuonitautien hoidossa. Tämän tutkimuksen tulokset viittaavat siihen, että sydämen endoteelisolut ovat erittäin muokkautuvia sekä fysiologisiin ärsykkeisiin että patologisiin stressitekijöihin. Nämä havainnot voivat auttaa kehittämään innovatiivisia ja uusia terapeuttisia mahdollisuuksia sydänsairauksien hoitoon

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

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

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 (pConclusions Short-term training improves adipose tissue metabolism both in healthy and IR participants independently of the sex. Adipose tissue angiogenesis and gene expression was only significantly affected in IR participants.</div

The SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells : Implications for COVID-19 vascular research

Humanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, so it is impor-tant to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the CNS, heart, and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed. In a number of other organs, pericytes do not express ACE2, including in the lung where ACE2 instead is expressed in bronchial epithelium and alveolar type II cells. The onset of ACE2 expression is organ specific: in bronchial epithelium already at birth, in brain pericytes before, andin heart pericytes after postnatal day 10.5. Establishing the vascular localization of ACE2 expression is central to correctly interpret data from modeling COVID-19 in the mouse and may shed light on the cause of vascular COVID-19 complications.Peer reviewe

Vascular Endothelial Growth Factor Receptor 1 regulating angiogenesis and cardiac growth in adult heart

The heart acts as a muscular pump in the hub of closed blood vascular circulatory loop to transport oxygenated blood and nutrients to various organs and collecting the deoxygenated blood back to the right ventricle for re-oxygenation in the lungs. Indigenously, the heart consists of two major cell populations; myocytes (cardiomyocytes) and non-myocytes (endothelial cells, vascular smooth muscle cells, pericytes, fibroblasts, leukocytes, pace maker cells and purkinje fibers) contributing to the structure and normal function of the heart. The presence of abundant endothelial cells, bi-directional paracrine communication between endothelial cells (ECs) and cardiomyocytes (CMCs), secretory properties of ECs regulating the heart growth, metabolism, maintaining blood vessel homeostasis and vascular tone further augments the indispensable role of cardiac ECs (Pinto, A.R. et al., 2016, Aird, W.C., 2007 and 2012). The Vascular Endothelial Growth Factors (VEGFs) and VEGF receptors (VEGFRs) play a crucial role in the development and maintenance of cardiovascular system. Previous findings have demonstrated the critical role of VEGFR1 during embryogenesis and its absence is embryonically lethal (Fong, G.H. et al., 1995; Fong, G.H. et al., 1999). Global deletion of VEGFR1 in adult mice, in turn, resulted in increased angiogenesis of various organs like heart, liver, lungs, kidney and brain (Ho, V.C. et al., 2012). However, the cell specific role (physiological and pathological) of VEGFR1 in the adult mouse heart remains unknown. In this study, I first assessed and validated the endothelial specificity of PdgfbCreERT2-Cre line in adult mice by crossing it with Td-Tomato reporter mouse line. Then we crossed PdgfbCreERT2 mice with VEGFR1 floxed mice, and after tamoxifen treatment, the recombination and deletion efficiency were analyzed in the heart. We also treated mice with AAV9-VEGF-B, which is a ligand for VEGFR1. Effects on cardiac vasculature and cardiomyocyte growth were analyzed. The findings of this thesis demonstrate that both deletion of endothelial VEGFR1 and overexpression of VEGF-B induce angiogenesis and modest cardiac hypertrophy in the heart. Mechanistically, this was mediated by activation of VEGFR2 pathway, as the effects were blocked by VEGFR2 antibody treatment. In conclusion, activation of VEGFR2 in cardiac endothelial cells by releasing its endogenous ligand VEGF from its decoy receptor VEGFR1 leads to angiogenesis and physiological cardiac hypertrophy

Cardiovascular disease risk factors induce mesenchymal features and senescence in mouse cardiac endothelial cells

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.Peer reviewe

STAT5b is a key effector of NRG-1/ERBB4-mediated myocardial growth

The growth factor Neuregulin-1 (NRG-1) regulates myocardial growth and is currently under clinical investigation as a treatment for heart failure. Here, we demonstrate in several in vitro and in vivo models that STAT5b mediates NRG-1/EBBB4-stimulated cardiomyocyte growth. Genetic and chemical disruption of the NRG-1/ERBB4 pathway reduces STAT5b activation and transcription of STAT5b target genes Igf1, Myc, and Cdkn1a in murine cardiomyocytes. Loss of Stat5b also ablates NRG-1-induced cardiomyocyte hypertrophy. Dynamin-2 is shown to control the cell surface localization of ERBB4 and chemical inhibition of Dynamin-2 downregulates STAT5b activation and cardiomyocyte hypertrophy. In zebrafish embryos, Stat5 is activated during NRG-1-induced hyperplastic myocardial growth, and chemical inhibition of the Nrg-1/Erbb4 pathway or Dynamin-2 leads to loss of myocardial growth and Stat5 activation. Moreover, CRISPR/Cas9-mediated knockdown of stat5b results in reduced myocardial growth and cardiac function. Finally, the NRG-1/ERBB4/STAT5b signaling pathway is differentially regulated at mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to control human subjects, consistent with a role of the NRG-1/ERBB4/STAT5b pathway in myocardial growth.peerReviewe

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