Project 4

Background and preliminary work: Aging is a major independent risk factor for the development and progression of cardiovascular diseases and is characterized by increased oxidative stress and low-grade inflammation. These conditions affect the vessel wall in all vascular beds leading to – among others – endothelial cell senescence ^{1, 2} and a phenotypic switch in vascular smooth muscle cells (VSMC) ³. Both these phenomena contribute to the known age-associated vascular dysfunction. Cellular senescence, a response to various stressors, is generally characterized by persistent cell cycle arrest, macromolecular damage, metabolic changes and a senescence-associated secretory phenotype ⁴, with the latter having an impact on the tissue environment of senescent cells. Senescent endothelial cells show additional, cell-type specific features, like loss of endothelial Nitric Oxide Synthase ^{5, 6} and their major anti-oxidative enzyme Thioredoxin-1 with concurrent upregulation of the ROS-producing enzyme NADPH oxidase 4 ⁷. Under physiological conditions, VSMC are quiescent and contractile to maintain the normal vessel structure and assure proper blood pressure regulation. In response to stress or injury as well as during the aging process, they adopt a less differentiated so-called synthetic phenotype. This switch encompasses decreased expression of contractile proteins and increased expression of pro-proliferative and migratory factors and secretion of large amounts of Collagen, Elastin and Matrix Metalloproteases causing remodeling of the extracellular matrix. The majority of the alterations involved in the induction of endothelial senescence and VSMC phenotypic switching is due to altered gene expression, i.e. transcriptome changes in both cell types.

The Haendeler group has demonstrated that multiple risk factors for cardiovascular diseases like aging and the low-grade inflammation, air pollution or unhealthy diet induce senescence in endothelial cells. The dysfunction associated with the senescent phenotype, manifested by e.g. reduced migratory capacity and increased apoptosis sensitivity, is caused by signaling alterations leading to a reduced NO bioavailability, lysosomal overactivation and increased oxidative stress ^5-11. Moreover, the group showed that senescence-induction with low-density lipoprotein leads to massive transcriptome changes ⁶ similar to those observed after treatment with concentrations of lipopolysaccharide inducing endothelial cell activation and apoptosis ¹².

The Sonkusare group demonstrated that Ca²⁺-signals in the endothelium are impaired in cardiovascular disorders causing increased vasoconstriction and blood pressure. One important contributor to that is the Transient Receptor Potential Vanilloid 4 channel (TRPV4) localized within myoendothelial projections, the portion of the arterial wall where endothelial cells and smooth muscle cells make direct contact. TRPV4, a critical Ca²⁺ influx pathway in endothelial cells, is downregulated during aging ¹³. Moreover, its function is impaired in hypertension ¹⁴ and by oxidative modifications ^{15, 16}, which occur under oxidative stress that is observed in aged tissues. Interestingly, the group showed that stimulation of α1-adrenergic receptors in VSMC activated endothelial TRPV4 channels and limited vasoconstriction ¹⁷. Moreover, also TRPV4 in VSMC participated in blood pressure regulation, and, depending on localization in different microdomains affected it positively or negatively ¹⁸. Finally, the detrimental effects of aging on endothelial Ca²⁺- regulation and vasodilation are not only due to downregulation of TRPV4, but also involved a diminished Calreticulin expression and increased ER stress ¹⁹.

However, it is unclear if the above-mentioned transitions in endothelial and smooth muscle cells during aging occur simultaneously or consecutively with changes in one cell type causing alterations in the other and if this happens in all vascular beds. Moreover, it has become clear that only a subpopulation of those cells adopts another phenotype. Up to now the reasons for this cellular heterogeneity have not been unraveled at all.

Hypothesis: We hypothesize that endothelial cell senescence induced by aging and/or high blood pressure precedes the smooth muscle cell transitions in the different vascular beds, with both cell types having a major impact on vascular function. To address the questions of the heterogeneous phenotypic changes and the sequence of events, we will combine our expertise on vascular senescence and redox signaling and on vascular physiology and pathophysiology as well as the role of TRPV4 in vivo.

Work program: As a prerequisite for understanding the mechanisms underlying age-associated changes in cellular plasticity and heterogeneity in the vascular wall, we will perform single-cell RNA sequencing (scRNA seq) in the facilities available in both locations (BMFZ, HHU Düsseldorf, Germany and Genome Analysis and Technology Core, School of Medicine, University of Virginia, Charlottesville, USA). For this, we will use tissue sections of the micro- and macrocirculation of adult and aged mice. In-depth bioinformatic analyses will be performed within the IRTG (P5, P10) and with the help of the Bioinformatics Core, School of Medicine, University of Virginia, Charlottesville, USA. The scRNAseq data will provide information about transcriptional signatures of senescent endothelial and synthetic smooth muscle cells and how the proportions of these cells change with increasing age. We expect differences not only with aging, but also in the different vascular beds and in animals with cell-specific TRPV4-deficiency, which are available in the American laboratory ^{16, 18}. These mice are characterized by high blood pressure, one of the main risk factors for atherosclerosis, and it is known that endothelial cell senescence as well as VSMC switching participate in the pathogenesis of the disease ^{20, 21}. Given the hypertensive phenotype of these animals, the data will also be compared to the ones from P3 (Stegbauer/Isakson), in which blood pressure is raised by high salt intake in wildtype mice.

Clusters of senescent endothelial and synthetic smooth muscle cells will be defined by the expression of cell type specific markers and the GO-terms “cellular senescence” and “protein secretion”; we will also include the term “cell migration”, as this process is reciprocally affected in senescent endothelial cells and synthetic VSMC as described above. To get an impression about the interrelation between senescence induction in the endothelium and phenotype switching in the muscular layer of the vascular wall we will identify precursor stages by trajectory inference. This information will at least provide clues about the sequence of events with respect to the alterations in both cell types and if they accumulate over time.

The transcriptome profiles will also allow conclusions with respect to pleiotropic effectors and signaling pathways, which coordinately regulate the transcription of multiple genes and thus, the phenotypic switches. Furthermore, we will get clues about the differences between the cell populations that are driven into senescence and phenotypic switching, respectively, and cells that do not adopt these fates including the signaling networks involved in these decisions.

After having identified such pleiotropic effectors, we will validate their impact on the induction and progression of endothelial cell senescence and VSMC switching ex vivo an in vivo. The ex vivo validation will be performed by the German graduate student as the Haendeler laboratory has long standing expertise in ex vivo models for endothelial cell senescence ^{5, 7, 9, 11} and can easily adopt techniques for VSMC switching from other partners in the IRTG, which have established these long time ago ²². Selected pleiotropic regulators involved in the decisions driving endothelial cells into senescence and VSMC into phenotypic switching or not, will be overexpressed or downregulated in primary human macro- and microvascular endothelial cells and vascular smooth muscle cells using lentiviral vectors. Then, senescence will be induced by oxidative stress or low-grade inflammation, and VSMC switching by treatment with oxidized phospholipids or TGF-β1. The expression of markers for senescence and the synthetic phenotype, respectively, will be monitored over time using real-time PCR, immunoblotting and immunofluorescence staining. The latter will also allow to estimate the percentage of senescent or phenotypically switched cells. Overall, these approaches will reveal, how the pleiotropic regulators defined on the basis of the bioinformatic analysis of the scRNAseq data, affect the phenotypic changes in both cell types qualitatively and quantitatively. All necessary techniques are established in the laboratory of the German PI ^{7, 11, 12, 23} or can be adopted as described above. During his/her stay in Germany, the American student will be trained in these models and participate in the ex vivo experiments.

In addition, we will setup co-culture models of endothelial and vascular smooth muscle cells to mimic, at least partially, the arterial wall, again with the help of one of the other American PIs in the IRTG, who has established such models to study myoendothelial junctions ²⁴. In such models it is possible to genetically modify or pretreat either of the two cell types. This would open up the possibility to address the question of which phenotypic alteration might precede or even induce the one in the other cell type, or if they occur independently of each other.

The in vivo aspect of the validation will be covered on the American side studying the pleiotropic regulators examined ex vivo in the Haendeler laboratory by refined fluorescence imaging techniques. Therefore, arterial vessels from different vascular beds from mice of different age, including animals with cell-specific TRPV4-deficiency, will be co-stained for these regulators, typical senescence markers like proteins from the Cyclin dependent Kinase Inhibitor family and proteins indicative of the contractile or synthetic smooth muscle phenotype. Although endothelial cells and vascular smooth muscle cells can be recognized topographically in the vascular wall, staining for cell-specific markers will be used for their unequivocal identification. These immunofluorescence analyses will be performed on cryosections, but also on en face preparations, which give a much better three-dimensional impression of larger regions of the vascular wall. All necessary techniques are routinely used in the Sonkusare laboratory and have already been applied in a previous collaboration between both PIs ²⁵. The Sonkusare laboratory also specializes in superresolution microscopy at 10 nm resolution. This technique allows visualization of single protein molecules and their colocalization with other interacting proteins in freshly isolated endothelial and smooth muscle cell ¹⁸. This will give the German graduate student the option to learn the en face preparations and super-resolution microscopy during the exchange phase and to work on this part of the project together with the American student.

In summary, this joint project will identify why only a fraction of endothelial and smooth muscle cells in the vascular wall is affected by the aging process, an independent risk factor for the development of cardiovascular diseases, and by high blood pressure, which promotes the onset and progression of atherosclerosis, an age-associated disease, which can result in fatal outcomes like myocardial infarction and stroke.

Added value of the collaboration: The complementary, non-redundant experimental models and scientific equipment as well as the different expertise of the PIs, create a synergism, from which both partner laboratories, and especially also the graduate students, will profit.