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ß1-integrins are essential for angiogenesis but the mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. BRAG2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of BRAG2 in EC and angiogenesis and the underlying molecular mechanisms remains unclear. siRNA-mediated BRAG2-silencing reduced EC angiogenic sprouting and migration. BRAG2-siRNA-transfection differentially affected a5ß1- and aVß3-integrin function: specifically, BRAG2-silencing increased focal/fibrillar adhesions and EC adhesion on ß1-integrin-ligands (fibronectin and collagen), while reducing the adhesion on the aVß3-integrin-ligand, vitronectin. Consistent with these results, BRAG2-silencing enhanced surface expression of a5ß1-integrin, while reducing surface expression of aVß3-integrin. Mechanistically, BRAG2 mediated recycling of aVß3-integrins and endocytosis of ß1-integrins and specifically of the active/matrix bound a5ß1-integrin present in fibrillar/focal adhesions (FA), suggesting that BRAG2 contributes to the disassembly of FA via ß1-integrin-endocytosis. Arf5 and Arf6 are promoting downstream of BRAG2 angiogenic sprouting, ß1-integrin-endocytosis and the regulation of FA. In vivo silencing of the BRAG2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitral injection of plasmids containing BRAG2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveals that BRAG2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating ß1-integrin internalization and associates for the first time the process of ß1-integrin endocytosis with angiogenesis.
Cardiovascular diseases are still regarded as the main cause of death in the modern world. However, the generic term "cardiovascular diseases" is not uniformly defined. It essentially describes diseases of the cardiovascular system and includes diseases such as hypertension, arteriosclerosis, myocardial infarctions, heart failure, coronary heart diseases, rheumatic heart diseases and heart valve defects. In addition to the well-known risk factors such as obesity, smoking, hypercholesterolemia and lack of exercise, age is a further risk factor that plays an important role in the development of cardiovascular diseases. As the modern societies age; this becomes an increasing problem.
But why does the prevalence of cardiovascular diseases increase with age? In gen-eral, age-dependent changes at the cellular level are assumed to be responsible for the pathological changes in the cardiac and vascular tissues. Important mechanisms such as autophagy, oxidative stress, mitochondrial dysfunctions, genomic instability, cellular senescence and disturbances in signaling pathways of growth factors play a decisive role. In old age, myocardial hypertrophy occurs, which results in cardiac wall thickening and an altered geometry of the ventricle. Chronic inflammations, paracrine and age-dependent cell-intrinsic factors further lead to activation of cardiac fibro-blasts with increase cell proliferation, collagen secretion and matrix cross-linking. The consequences are interstitial and perivascular fibrosis, which stiffen the heart and blood vessels. Oxidative stress and inflammations additionally attack the blood ves-sels and impair endothelial function, which is further aggravated by possible pre-existing conditions such as diabetes mellitus and hypertension.
In the past decades, the main focus has therefore been on researching these age-dependent changes in the hope of better understanding cardiovascular ageing and developing possible regenerative interventions. By studying the repair mechanisms of other organs such as the lungs and the bone marrow, the endothelium in particular showed a high regenerative capacity, which influences the proliferation and cell func-tion of the surrounding cells.
For a long time, the general opinion was that the endothelium is only the internal lin-ing of blood and lymphatic vessels, as well as the heart chambers, which as a single-layer barrier guarantees the integrity of the blood vessels. However, endothelial cells are very heterogeneous, depending on the type of blood vessel and the type of tis-sue they serve. In addition to their barrier function, endothelial cells also regulate the exchange of substances between blood and tissue, stimulate the formation of new blood vessels and re-model existing vascular networks. They are also able to re-structure the extracellular matrix that surrounds them. They release not only matrix proteins, but also cytokines and growth factors into the extracellular space. On de-mand, these factors are then released and stimulate angiogenesis or cell prolifera-tion. In addition, the secretion of various matrix proteins not only stabilizes the cellu-lar neighborhood, but also regulates various cell functions.
By modelling the endothelial environment - the so-called vascular niche - endothelial cells are able to communicate with the surrounding cells. As a result, a regenerative effect of the vascular niche has already been described in various organs. In the liv-er, for example, it has been shown that increased concentrations of endothelial Ang2 and decreased endothelial activin A after partial hepatectomy stimulate the prolifera-tion of hepatocytes and thus liver regeneration. In the bone marrow, endothelial cells mobilize stem cells via nitric oxide and in the lungs, endothelial MMP14 releases growth factors from the extracellular matrix, which stimulate epithelial cell prolifera-tion after partial pneumectomy. Whether such a regenerative effect of the vascular niche also plays a role in the heart is largely unknown.
Since both the regenerative capacity of the heart and endothelial function decrease with age, the aim of this dissertation was to investigate the role of the vascular niche and endothelial cell communication in the aged heart. Human cell lines as well as mouse and artificial rat models were used for these investigations. Since this thesis is a cumulative dissertation with partially published papers, it is divided into three parts.
In the first part of this thesis, the transcriptional signature of secretory genes in the aged cardiac endothelium was studied. Perfused endothelial cells from hearts of young (12-week-old animals) and old mice (20-month-old animals) were isolated and used for bulk RNA sequencing. The two matrix proteins laminin β1 and β2 were among the top-regulated genes. While laminin β2 was particularly expressed in the young cardiac endothelium, laminin β1 was predominantly found in the old endotheli-um. This change in laminin expression was confirmed histologically at protein level and its autocrine function was investigated in vitro. To mimic the in vivo situation in vitro, cell culture dishes were coated with human recombinant laminin 421 or laminin 411 and sutured with human endothelial cells from the umbilical vein (HUVEC). Di-verse functional investigations showed that endothelial cells migrated and adhered poorly in the presence of laminin 411, while in Matrigel tube formation assays HU-VEC formed reduced endothelial networks when cultured on LM 411.
...
One of the key functions of blood vessels is to transport nutrients and oxygen to distant tissues and organs in the body. When blood supply is insufficient, new vessels form to meet the metabolic tissue demands and to re-establish cellular homeostasis. Expansion of the vascular network through sprouting angiogenesis requires the specification of ECs into leading (sprouting) tip and following (non-sprouting) stalk cells. Attracted by guidance cues tip cells dynamically extend and retract filopodia to navigate the nascent vessel sprout, whereas trailing stalk cells proliferate to form the extending vascular tube. All of these processes are under the control of environmental signals (e.g. hypoxia, metabolism) and numerous cytokines and peptide growth factors. The Dll4/Notch pathway coordinates several critical steps of angiogenic blood vessel growth. Even subtle alterations in Notch activity can profoundly influence endothelial cell behavior and blood vessel formation, yet little is known about the intrinsic regulation and dynamics of Notch signaling in endothelial cells. In addition, it remains an open question, how different growth factor signals impinging on sprouting ECs are coordinated with local environmental cues originating from nutrient-deprived, hypoxic tissue to achieve a balanced endothelial cell response. Acetylation of lysines is a critical posttranslational modification of histones, which acts as an important regulatory mechanism to control chromatin structure and gene transcription. In addition to histones, several non-histone proteins are targeted for acetylation reversible acetylation is emerging as a fundamental regulatory mechanism to control protein function, interaction and stability. Previous studies from our group identified the NAD+-dependent deacetylase SIRT1 as a key regulator of blood vessel growth controlling endothelial angiogenic responses. These studies revealed that SIRT1 is highly expressed in the vascular endothelium during blood vessel development, where it controls the angiogenic activity of endothelial cells. Moreover, in this work SIRT1 has been shown to control the activity of key regulators of cardiovascular homeostasis such as eNOS, Foxo1 and p53. The present study describes that SIRT1 antagonizes Notch signaling by deacetylating the Notch intracellular domain (NICD). We showed that loss of SIRT1 enhances DLL4-induced endothelial Notch responses as assessed by different luciferase responsive elements as well as transcriptional analysis of Notch endogenous target genes activation. Conversely, SIRT1 gain of function by overexpression of pharmacological activation decreases induction of Notch targets in response to DLL4 stimulation. We also showed that the NICD can be directly acetylated by PC AF and p300 and that SIRT1 promotes deacetylation of NICD. We have identified 14 lysines that are targeted for acetylation and their mutation abolishes the effects of SIRT1 of Notch responses. Furthermore, over-expression or activation of SIRT1 significantly reduces the levels of NICD protein. Moreover, SIRT1-mediated NICD degradation can be reversed by blockade of the proteasome suggesting a mechanism resulting from ubiquitin-mediated proteolysis. Indeed, we have shown that SIRT1 knockdown or pharmacological inhibition decreased NICD ubiquitination. We propose a novel molecular mechanism of modulation of the amplitude and duration of Notch responses in which acetylation increases NICD stability and therefore permanence at the promoters, while SIRT1, by inducing NICD degradation through its deacetylation, shortens Notch responses. In order to evaluate the physiological relevance of our findings we used different models in which the Notch functions during blood vessel formation have been extensively characterized. First, retinal angiogenesis in mice lacking SIRT1 activity shows decreased branching and reduced endothelial proliferation, similar to what happens after Notch gain of function mutations. ECs from these mice exhibit increased expression of Notch target genes. Second, these results were reproducible during intersomitic vessel growth in sirt1-deficient zebrafish. In both models, the defects could be partially rescued by inhibition of Notch activation. Third, we used an in vitro model of vessel sprouting from differentiating embryonic bodies in response to VEGF in a collagen matrix. Our results showed that Sirt1-deficient cells shows impaired sprouting which correlated with increased NICD levels. In addition, when in competition with wild-type cells in this assay, Sirt1-deficient cells are more prone to occupy the stalk cell position. Taken together, our study identifies reversible acetylation of NICD as a novel molecular mechanism to adapt the dynamics of Notch signaling and suggest that SIRT1 acts as a rheostat to fine-tune endothelial Notch responses. The NAD+-dependent feature of SIRT1 activity possibly links endothelial Notch responses to environmental cues and metabolic changes during nutrient deprivation in ischemic environments or upon other cellular stresses.
Cardiovascular disease is the leading cause of death worldwide. Aging is among the greatest risk factors for cardiovascular disease. Cardiovascular disease comprises several diseases, for example myocardial infarction, elevated blood pressure and stroke. Many processes are known to promote or worsen cardiovascular disease and in the present study, cellular senescence and inflammatory activation were of special interest, as they have a strong association to aging and can be seen as hallmarks of cellular aging.
Long noncoding RNAs (lncRNAs) are noncoding RNAs with a length of more than 200 nucleotides. In recent years, numerous regulatory functions were shown for these transcripts and lncRNAs were shown to directly interact with DNA, RNA and proteins. The long noncoding RNA H19 was among the first described noncoding RNAs and was initially shown to act as a tumor suppressor. More recently, several studies showed oncogenic roles for H19. In regards to the cardiovascular system, H19 was not analyzed before.
We show that H19 is the most profoundly downregulated lncRNA in endothelial cells of aged mice compared to young littermates. Microarray analysis of human primary endothelial cells upon pharmacological H19 depletion revealed an involvement of H19 in cell cycle regulation. Loss of H19 in human endothelial cells in vitro led to reduced proliferation and to increased senescence. H19 depletion was shown to counteract proliferation before, but none of the described mechanisms applied to endothelial cells. We show that the reduction in proliferative capacity and the pro-senescent function of H19 is most probably mediated by an upregulation of p16ink4A and p21 upon H19 depletion.
When we compared the angiogenic capacity of aortic endothelial cells from young and aged mice in an aortic ring assay, rings from aged mice showed a reduced cumulative sprout length. Interestingly, pharmacological inhibition of H19 in aortic rings of young animals, where H19 is highly expressed, was sufficient to reduce the cumulative sprout length to levels we observed from aged animals. Furthermore, overexpression of human H19 in aortic rings of aged mice, where H19 is poorly expressed, rescued the impaired angiogenic capacity of aged endothelial cells.
We generated inducible endothelial-specific H19 knockout mice (H19iEC-KO) and subjected these animals to hind limb ischemia surgery followed by perfusion analysis in the hind limbs by laser-doppler velocimetry and histological analysis. Perfusion in the operated hind limb was increased in H19iEC-KO compared to Ctrl littermates, which was in contrast to a reduction in capillary density in the operated hind limbs of H19iEC-KO animals compared to Ctrl littermates and to our previous results. Analysis of arteriogenesis revealed an increase in collateral growth upon EC-specific H19 depletion in the ischemic hind limbs, which explains the increase in perfusion despite the reduction in capillary density. Further characterization of the animals revealed an increase in leukocyte infiltration into the tissue in the ischemic hind limbs upon endothelial-specific H19 depletion, indicating a potential role of H19 in inflammatory tissue activation.
Reanalysis of the microarray data from human primary endothelial cells upon H19 depletion revealed an association of H19 with inflammatory signaling and more specifically with IL-6/JAK2/STAT3 signaling. Analysis of cell surface adhesion molecule expression revealed an upregulation of ICAM-1 and VCAM-1 on mRNA level and an increase of the abundance of the two proteins on the cell surface of human primary endothelial cells. Consequently, adhesion of isolated human monocytes to human primary endothelial cells was increased upon H19 depletion in vitro. Interestingly, TNF-α mediated inflammatory activation of primary human endothelial cells repressed H19 expression. H19 did not function via previously described mechanisms. We excluded a competitive endogenous RNA (ceRNA) function for H19 in endothelial cells and showed that miR-675, which is processed from H19, does not play a role in the endothelium. Furthermore, H19 did not regulate previously described genes or pathways.
Analysis of transcription factor activity upon H19 depletion and overexpression revealed a differential activity of STAT3. STAT3 phosphorylation at TYR705 and thus activation was increased upon H19 depletion. Inhibition of STAT3 activation using a small compound inhibitor abolished the effects of H19 depletion on mRNA expression of p21, ICAM-1 and VCAM-1 and on proliferation, indicating that the effects of H19 are at least partially mediated via STAT3. STAT3 was shown to have positive effects on the cardiovascular system before, most likely due to upregulation of VEGF in a STAT3-dependent manner. We were not able to confirm previously described mechanisms for STAT3 in the present study and propose a new mechanism of action for the H19-dependent regulation of STAT3. Taken together, these results identify the long noncoding RNA H19 as a pivotal regulator of endothelial cell function. Figure 38 summarizes the described functions of H19 in endothelial cells.
Flow hemodynamics regulates endothelial cell (EC) responses and laminar shear stress induces an atheroprotective and quiescent phenotype. The flow-responsive transcription factor KLF2 is a pivotal mediator of endothelial quiescence, but the precise mechanism is unclear. In this doctoral study, we assessed the hypothesis that laminar shear stress and KLF2 regulate endothelial quiescence by controlling endothelial metabolism.
Laminar flow exposure and KLF2 over expression in HUVECs reduced glucose uptake. Endothelial specific deletion of KLF2 (EC-KO) in mice and subsequent infusion of labeled glucose in Langendorff perfused hearts induced glucose uptake in ECs lacking KLF2. Bioenergetic measurements revealed that KLF2 reduces and glycolytic acidification in vitro.
Mechanistically, RNA sequencing analysis of shear stimulated ECs showed reduced expression of key glycolytic enzymes Hexokinase 2, PFKFB3 and PFK-1. KLF2 also reduced expression of these enzymes at protein level. KLF2 knockdown in shear stimulated ECs reversed the reduction in expression of PFKFB3 and PFK-1, indicating KLF2-dependency. Promoter analysis revealed KLF binding sites in the promoter of PFKFB3 and KLF2 over expression markedly reduced PFKFB3 promoter activity which was abolished on mutation of the KLF binding site. In addition, PFKFB3 knockdown reduced glycolysis while over expression increased glycolysis. Over expression of PFKFB3 along with KLF2 partially reversed the KLF2-mediated reduction in glycolysis. Importantly, PFKFB3 over expression reversed KLF2-mediated reduction in angiogenic sprouting and network formation in vitro. Ex-vivo aortic ring assays revealed an increase in endothelial sprouting from aortas from KLF2 EC-KO mice, which was partially reversed upon PFKFB3 inhibition by 3-PO.
In conclusion, work performed during this doctoral thesis demonstrates that laminar shear stress and KLF2 mediated repression of endothelial metabolism via regulation of PFKFB3 contributes to the anti-angiogenic and quiescent properties of the endothelium.
Kardiovaskuläre Erkrankungen nehmen einen großen Sektor des gegenwärtigen Krankheitsspektrums ein. Die Entdeckung von Stammzellen, die sich zu Gefäßen oder Herzmuskelzellen entwickeln können, bietet neben bereits etablierten Behandlungen völlig neue therapeutische Ansatzpunkte zur kardialen Regeneration dieser Patienten. Neben embryonalen oder adulten Stammzellen kommen auch leicht aus dem peripheren Blut zu gewinnende endotheliale Vorläuferzellen für mögliche Therapien in Frage. Um den Ansatz der Differenzierung von Stamm- oder Vorläuferzellen in Herzmuskelzellen in vitro zu untersuchen, wurde ein bereits bekanntes Modell der Ko-Kultur von neonatalen Rattenkardiomyozyten mit verschiedenen Populationen von Stamm- oder Vorläuferzellen genutzt. Anlehnend an dieses Modell wurden in dieser Arbeit EPCs für sechs Tage zusammen mit neonatalen Kardiomyozyten der Ratte kultiviert. Es zeigt sich, dass EPCs nach sechs Tagen Ko-Kultur mit neonatalen Rattenkardiomyozyten in der Lage sind, zu Kardiomyozyten zu differenzieren und typische kardiomyozytäre Eigenschaften aufweisen, zu denen beispielsweise die Expression kardiospezifischer Proteine gehören sowie die Integration mit umliegenden Kardiomyozyten. Nach Etablierung dieses Versuchsansatzes wurde die Differenzierungskapazität der EPCs KHK erkrankter Patienten untersucht, sowie der Einfluß von Statineinnahme der Patienten, da eine prinzipielle Wirkung der Statine auf EPCs bereits vielfach beschrieben wurde. Es zeigt sich, dass auch EPCs von KHK-Patienten in der Lage sind, zu Kardiomyozyten zu differenzieren, wobei eine verringerte Differenzierungsrate zu beobachten ist. Durch Behandlung der Patienten mit Statinen lässt sich diese verringerte Kapazität verbessern, wie sich nicht nur in einer Querschnittsuntersuchung, sondern auch im prospektiven Verlauf gezeigt hat. Der Mechanismus, über den Statine eine Verbesserung der EPC-Differenzierung erreichen, ist nicht geklärt. Interessanterweise sind Statine in vitro nicht in der Lage, die EPCDifferenzierung zu Kardiomyozyten zu verbessern. Der vielversprechende Ansatz der Regeneration von Gewebe durch Stammzellen wird durch die vergleichsweise geringe Ausbeute an differenzierten Zellen limitiert. Aus diesem Grunde wurden verschiedene Versuche durchgeführt, die Differenzierungsrate in vitro anzuheben. Leider zeigen VEGF (beschrieben ist beispielsweise ein positiver Effekt auf Überleben und Migration), 5’-Azacytidine (Erhöhung der Differenzierungsrate embryonaler Stammzellen) oder hypoxisch-konditioniertes Medium (Erhöhung der Differenzierung von Stammzellen in neurales Gewebe) keinen positiven Effekt auf die EPC-Differenzierungsrate zu Kardiomyozyten. Von grundlegender Bedeutung ist es, die Mechanismen der Differenzierung von Stamm- oder Vorläuferzellen aufzuklären. Erschwert wird diese Aufgabe durch die Möglichkeit, dass in verschiedenen Geweben verschiedene Mechanismen (Zellfusion auf der einen und Transdifferenzierung auf der andere Seite) für die Stammzellintegration verantwortlich sein könnten. Mithilfe einer Ko-Kultur der EPCs mit fixierten Kardiomyozyten konnte gezeigt werden, dass die Differenzierung der EPCs zu Kardiomyozyten den direkten Kontakt zu anderen Kardiomyozyten benötigt, jedoch nicht zwingend auf einer Zellfusion basiert. Um den Differenzierungsprozess weiter zu untersuchen, wurden die für die Zell-Zell- oder Zell-Matrix- Interaktion wichtigen Proteine untersucht. In einem Blockierungsversuch der für die Zell-Matrix- Interaktion wichtigen Integrine, ließ sich kein Nachweis für eine Rolle der Integrine für den Differenzierungsprozess erbringen. Für die Zell-Zell- Interaktion stellen die kalziumabhängigen Cadherine eine wichtige Gruppe dar. In einer Ko-Kultur, die in einem kalziumfreien Medium durchgeführt wurde, ließ sich eine signifikante Reduktion des Überlebens der EPCs feststellen. Ein weiterer Versuch, der mit einer Mischung verschiedener Cadherin-blockierender Antikörper durchgeführt wurde, zeigt eine signifikante Reduktion der Differenzierung der EPCs in Kardiomyozyten. Die Untersuchung, welches Cadherin für den Differenzierungsprozess eine besondere Bedeutung spielt, ist Gegenstand gegenwärtiger Untersuchungen. Diese Doktorarbeit zeigt, dass EPCs prinzipiell in der Lage sind, in Kardiomyozyten zu differenzieren. Ebenso sind EPCs KHK-erkrankter Patienten in der Lage in Kardiomyozyten zu differenzieren, jedoch zu einem geringeren Prozentsatz. Statinbehandlung steigert den Prozentsatz der EPC-Differenzierung bei KHK-erkrankten Patienten. Mit medikamentöser Behandlung (beispielsweise Statineinnahme) könnte der Stammzelltherapieansatz bei KHK-erkrankten Patienten unterstützt werden. Erste Hinweise für den komplexen Prozess der Progenitorzelldifferenzierung weisen auf einen kalziumabhängigen, Cadherin-vermittelten Mechanismus hin.
Zusammenfassend lässt sich sagen, dass eine medikamentöse Therapie mit Atorvastatin bei Patienten mit stabiler KHK zur Steigerung kultivierter EPCs mit verbesserter funktioneller Aktivität führt. Die Daten zeigen des weiteren, dass die Statintherapie nicht die Zahl hämatopoetischer Progenitorzellen erhöht, sondern die Differenzierung in zirkulierende EPCs fördert. Ein Faktor, wie z.B. VEGF, GM-CSF oder TNF-alpha, der die erhobenen Ergebnisse reflektiert bzw. vermittelt, konnte nicht gefunden werden. Allerdings konnte gezeigt werden, dass Atorvastatin über den PI3K-Signaltransduktionsweg, unabhängig von NO, die Differenzierung von EPCs stimuliert. In einer zweiten Studie konnte gezeigt werden, dass auch der ACE-Inhibitor Ramipril vor allem eine Verbesserung der funktionellen Aktivität der EPCs induzierte und ebenfalls zu einer Steigerung der Zahl der kultivierten EPCs führte. Aufgrund der starken Schwankungen der FACS-Messungen bei kleinen Patientenkollektiven besteht eine Diskrepanz zwischen den kultivierten und zirkulierenden EPCs. Auch konnte gezeigt werden, dass die EPC-Zahl und -Funktionalität vor Therapie durch den HGF-Serumspiegel reflektiert wurde und positiv mit ihm korrelierte. Diese Korrelation blieb jedoch unter Ramipriltherapie nicht bestehen, so dass davon auszugehen ist, dass der Einfluss von Ramipril nicht durch HGF, sondern über einen noch zu untersuchenden Mechanismus vermittelt wird. So können Statine und potentiell einige Subgruppen der ACE-Inhibitoren neue Therapieoptionen der KHK eröffnen.
Cardiovascular disease (CVD) is the leading cause of death in the western world. Aging as the major risk factor for the development of CVD leads to structural changes in the heart and the vasculature. In addition to endothelial cells, mural cells, including smooth muscle cells and pericytes, form the vascular wall. Pericytes are defined as the perivascular cells located in the basement membrane of the capillaries, which are the smallest components of the vascular system and ensure the gas exchange in the tissue. In the different parts of the terminal vascular bed, pericytes receive different phenotypes and organ-specific functions. In addition to the stabilization of the vascular wall, pericytes are relevant for the formation of new vessels. Due to their potential of multipotent stem cells, pericytes can differentiate into different cell types and thus take a position in developmental processes. Pericytes play a crucial role in the development and diseases of the vascular system. Moreover, pericyte coverage is reduced in the aged heart. Nonetheless, the function of pericytes in the heart and their importance during cardiac aging is not completely understood.
To study the pericyte population in the aging heart, we have performed single-nucleus RNA-sequencing analysis comparing hearts from 12-weeks-old (young) and 18-month-old (old) mice. The detailed analysis of 336 differentially expressed genes (DEG) revealed that Rgs5 is downregulated in aged pericytes. Regulator of G-protein signaling 5 (RGS5), an established marker for pericytes, is involved the regulation of the blood pressure and in the formation of various cardiovascular diesases, including cardiac hypertrophy, myocardial infarction and atherosclerosis. We have furthermore confirmed this observation in vivo. Gene ontology (GO) analysis of DEG revealed that aged pericytes are characterized by the downregulation of genes involved in cell adhesion. Further, we have performed cell biology approaches using human brain vascular pericytes (hBVP) to investigate the role of Rgs5 in pericytes in vitro. Efficient knockdown of RGS5, although has no effect on cellular metabolism, viability and endothelial permeability, induces a reduction of pericyte adhesion to both a gelatine matrix and endothelial cells in a 3D matrigel culture. This was associated with the formation of filopodia. The altered phenotype suggested a changing identity of the pericytes. We could confirm that a loss of RGS5 causes a decreased expression of the pericyte markers PDGFRb and NOTCH3 and also leads to an overexpression of COL1A1, a fibroblast marker.
Together, our findings suggest that RGS5 is required for pericyte adhesion to endothelial cells and its downregulation in the aged mural cells could explain the reduction of pericyte coverage in the aged hearts. Further, RGS5 may be the key regulator for pericyte identity, as pericytes show an altered expression profile of cellular markers. The dedifferentiation of pericytes to a more fibroblast-like cell type could explain the increased fibrosis during age-related cardiac remodeling. We believe that RGS5 is a great candidate to explore and study the molecular mechanisms that regulate pericyte function in the heart, both in homeostasis and during aging.
Chronische Herzinsuffizienz ist eine der führenden Todesursachen im Rahmen kardiovaskulärer Erkrankungen und ist mit einer hohen Anzahl an Komorbiditäten assoziiert. Unter anderem führt Herzinsuffizienz zu Veränderungen des Immunsystems, welche denen der Immunoseneszenz ähneln. Als einflussreiche Modulatoren ganzer molekularbiologischer Regelkreise sind microRNAs in den letzten Jahren zunehmend in den Fokus gerückt. Diese nicht-kodierenden, kurzen RNA-Einzelstränge können die Genexpression von vielen Zielgenen durch spezifisches Binden der jeweiligen mRNA Transkripte kontrollieren. Aufgrund zum Teil gewebe-, zelltyp- und prozess-spezifischer Expression können microRNAs auch als mögliche Biomarker für spezifische klinische Fragestellungen dienen.
Im Rahmen der vorliegenden Arbeit wurde die Expression von immunmodulatorischen microRNAs im peripheren Blut (PB) von jungen und gealterten gesunden Probanden (y/h bzw. o/h) sowie Patienten mit chronischer Herzinsuffizienz (CHF) untersucht. Dabei wurde ein Bezug auf Immunoseneszenz bzw. die Auswirkung von CHF auf das Immunsystem hergestellt. Im Rahmen dessen wurden Leukozyten-, insbesondere Lymphozyten-Subpopulationen analysiert.
Hierzu wurden Probanden in die drei folgenden Gruppen eingeschlossen: Patienten mit CHF (n=18, durchschnittliches Alter 64 Jahre), alters-korrelierte gesunde Probanden (n=13, durchschnittliches Alter 64 Jahre) sowie junge gesunde Probanden (n=30, durchschnittliches Alter 25 Jahre). Neben der Erhebung der klinischen Daten wurde peripheres Blut zur Bestimmung der microRNA-Expressionslevels sowie für durchflusszytometrische Analysen der Leukozytenpopulationen gewonnen.
In den Expressionsanalysen konnte eine alters- und herzinsuffizienz-abhängige Dysregulation einzelner microRNAs beobachtet werden. Insbesondere Mitglieder der miR-181-Familie, spezifisch miR-181a und miR-181c, waren im Alter niedriger exprimiert, zudem war die Expression von miR-181c bei Vorliegen einer CHF deutlicher reduziert. Des Weiteren zeigte sich eine altersabhängige erhöhte Expression von miR-34a, wobei das Vorliegen von CHF keine Auswirkung auf die Expression zeigte. Bei den microRNAs miR-146a und miR-223 konnte keine signifikante alters- oder CHF-abhängige Regulation nachgewiesen werden. Lediglich zeigte bei der miR-155 zeigte sich eine signifikante Reduktion bei Vorliegen einer CHF im Vergleich o/h Probanden.
In Hinblick auf die Leukozytenpopulationen im peripheren Blut wiesen Patienten mit CHF höhere Zahlen an Leukozyten auf, alle miR-181 Mitglieder zeigten hierbei eine inverse Korrelation. Dagegen stellte sich in Hinblick auf die Zusammensetzung der Leukozyten-Subpopulationen eine Reduktion der Lymphozytenfraktion im Alter dar, besonders bei Patienten mit CHF. Insbesondere zeigte sich eine altersabhängige Abnahme der B-Lymphozytenpopulation, wobei auch hier das Vorliegen einer CHF diesen Effekt verstärkte. Die Expression miR-181a und miR-181c sowie miR-146a und miR-223 korrelierte positiv mit dem Anteil der B-Lymphozyten. Innerhalb der B-Zellen zeigte sich eine alters- und CHF-abhängige Reduktion der naïven B-Zellen, welche positiv mit der Expression von miR-181c, miR-146a und miR-223 korrelierte. Die beobachteten Veränderungen der B-Zell-Subpopulationen zeigten sich insbesondere bei CHF Patienten mit ischämischer Ursache im Vergleich zur dilatativen Kardiomyopathie.
Bei den untersuchten T-Lymphozyten-Subpopulationen zeigte sich eine altersabhängiger Abfall bei den zytotoxischen T-Zellen. Das Vorliegen einer CHF verstärkte diesen Effekt. Die beobachteten Veränderungen der T-Zell-Subpopulation korrelierten nicht mit der Expression der untersuchten microRNAs.
Im Gegensatz zu den lymphoiden Subpopulationen zeigte sich ein Anstieg der neutrophilen Granulozyten und der Monozyten im Alter, es stellte sich jeweils eine negative Korrelation mit der Expression von miR-181 Transkripten sowie miR-155, miR-146a und miR-223 dar.
Zusammenfassend zeigten sich signifikant erniedrigte Expressionslevels von miR-181c im Alter, was mit Immunoseneszenz-bedingten Veränderungen des peripheren Bluts einherging. Diese Veränderungen zeigten sich zusätzlich verstärkt bei Patienten mit CHF. Zukünftig könnten miR-181c Expressionslevel in peripherem Blut als Biomarker für die Immunfunktionen bei CHF Patienten dienen und in Hinblick auf eine mögliche prospektive Information evaluiert werden.
Investigation of the in vivo function of two adhesion GPCRs : GPR116 (ADGRF5) and ELTD1 (ADGRL4)
(2015)
GPR116 and ELTD1 are two adhesion GPCRs from different subgroups, which had been reported to be expressed in micro-vascular endothelia. To investigate GPR116 and ELTD1 in vivo functions, I first generated mice, which express mCherry under the control of the Gpr116 promoter. I was able to confirm GPR116 expression in endothelial cells of microvessels and also in alveolar epithelial type II cells of the lung using mCherry reporter mice. Co-expression of GPR116 with an endothelial cell marker could already be seen at embryonic day 12.5. To further study ELTD1 expression in vivo, knock-in LacZ reporter mice were obtained, in which the whole coding region of Eltd1 had been replaced by the cDNA encoding ß-galactosidase through homology recombination. I found Eltd1 to be expressed in vessels of different organs already at embryonic day 9.5. In addition, GPCR expression data on isolated cells showed that GPR116 and ELTD1 are also highly expressed in cardiomyocytes and adipocytes, but not in smooth muscle cells. Subsequently, I generated GPR116 and ELTD1 knockout mouse lines. Surprisingly, the global knockout of either GPR116 or ELTD1 alone had no obvious effect on developmental angiogenesis or on the homeostasis of the mature vasculature. Considering a potential genetic redundancy, I crossed GPR116 and ELTD1 single knockout mice to generate GPR116 and ELTD1 double knockout animals. Mice lacking both, GPR116 and ELTD1, showed to various degrees of developmental defects of aortic arch arteries and the cardiac outflow tract, leading to the perinatal death of some mice and to a shortened lifespan of others which, in addition, showed cardiac hypertrophy. Furthermore, double knockout mice exhibited significant growth impairment at an early age and developed renal glomerulopathy with massive proteinuria as well as splenomegaly. Unexpectedly, endothelial cell- and cardiomyocyte-specific double knockout mice did not recapitulate any phenotype of the global double knockout mice. Concerning the expression of both receptors in adipocytes, the adipocytespecific double knockout mice were kept on high-fat diet. The results showed that GPR116 and ELTD1 in adipocytes are not involved in the regulation of adipose tissue size and glucose homeostasis. In conclusion, I could demonstrate that GPR116 is expressed in endothelial cells and in lung alveolar epithelial type II cells, while ELTD1 is expressed in the capillaries of various organs including heart, liver and lungs, small and intermediate vessels of the brain and pancreas as well as glomerular and intertubular vessels in kidneys. Both receptors are expressed already during embryonic development. Loss of Gpr116 and Eltd1 leads to developmental defects of aortic arch arteries and the cardiac outflow tract. No aberrant phenotype could be detected in single KO mice, indicating that loss of one receptor can be compensated by the other. A most important subject of further investigations will be to work out in which cell type GPR116 and ELTD1 contribute to the vascular and renal phenotypes observed in global double knockout mice.