Endothelial Cell Orientation and Polarity Are Controlled by Shear Stress and VEGF Through Distinct Signaling Pathways.

Vascular networks form, remodel and mature under the influence of multiple signals of mechanical or chemical nature. How endothelial cells read and interpret these signals, and how they integrate information when they are exposed to both simultaneously is poorly understood. Here, we show using flow-induced shear stress and VEGF-A treatment on endothelial cells in vitro, that the response to the magnitude of a mechanical stimulus is influenced by the concentration of a chemical stimulus, and vice versa. By combining different flow levels and different VEGF-A concentrations, front-rear polarity of endothelial cells against the flow direction was established in a flow and VEGF-A dose-response while their alignment with the flow displayed a biphasic response depending on the VEGF-A dose (perpendicular at physiological dose, aligned at no or pathological dose of VEGF-A). The effect of pharmaceutical inhibitors demonstrated that while VEGFR2 is essential for both polarity and orientation establishment in response to flow with and without VEGF-A, different downstream effectors were engaged depending on the presence of VEGF-A. Thus, Src family inhibition (c-Src, Yes, Fyn together) impaired alignment and polarity without VEGF-A while FAK inhibition modified polarity and alignment only when endothelial cells were exposed to VEGF-A. Studying endothelial cells in the aortas of VEGFR2Y949F mutant mice and SRC iEC-KO mice confirmed the role of VEGFR2 and specified the role of c-SRC in vivo. Endothelial cells of VEGFR2Y949F mutant mice lost their polarity and alignment while endothelial cells from SRC iEC-KO mice only showed reduced polarity. We propose here that VEGFR2 is a sensor able to integrate chemical and mechanical information simultaneously and that the underlying pathways and mechanisms activated will depend on the co-stimulation. Flow alone shifts VEGFR2 signaling toward a Src family pathway activation and a junctional effect (both in vitro and in vivo) while flow and VEGF-A together shift VEGFR2 signaling toward focal adhesion activation (in vitro) both modifying cell responses that govern orientation and polarity.

Primary cilia sensitize endothelial cells to BMP and prevent excessive vascular regression.

Blood flow shapes vascular networks by orchestrating endothelial cell behavior and function. How endothelial cells read and interpret flow-derived signals is poorly understood. Here, we show that endothelial cells in the developing mouse retina form and use luminal primary cilia to stabilize vessel connections selectively in parts of the remodeling vascular plexus experiencing low and intermediate shear stress. Inducible genetic deletion of the essential cilia component intraflagellar transport protein 88 (IFT88) in endothelial cells caused premature and random vessel regression without affecting proliferation, cell cycle progression, or apoptosis. IFT88 mutant cells lacking primary cilia displayed reduced polarization against blood flow, selectively at low and intermediate flow levels, and have a stronger migratory behavior. Molecularly, we identify that primary cilia endow endothelial cells with strongly enhanced sensitivity to bone morphogenic protein 9 (BMP9), selectively under low flow. We propose that BMP9 signaling cooperates with the primary cilia at low flow to keep immature vessels open before high shear stress-mediated remodeling.

YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development.

Formation of blood vessel networks by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How endothelial cells arise in adequate numbers and arrange suitably to shape functional vascular networks is poorly understood. Here we show that YAP/TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP/TAZ increase the turnover of VE-Cadherin and the formation of junction associated intermediate lamellipodia, promoting both cell migration and barrier function maintenance. This is achieved in part by lowering BMP signalling. Consequently, the loss of YAP/TAZ in the mouse leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP/TAZ integrate mechanical signals with BMP signaling to maintain junctional compliance and integrity whilst balancing endothelial cell rearrangements in angiogenic vessels.

A map of human circular RNAs in clinically relevant tissues.

Cellular circular RNAs (circRNAs) are generated by head-to-tail splicing and are present in all multicellular organisms studied so far. Recently, circRNAs have emerged as a large class of RNA which can function as post-transcriptional regulators. It has also been shown that many circRNAs are tissue- and stage-specifically expressed. Moreover, the unusual stability and expression specificity make circRNAs important candidates for clinical biomarker research. Here, we present a circRNA expression resource of 20 human tissues highly relevant to disease-related research: vascular smooth muscle cells (VSMCs), human umbilical vein cells (HUVECs), artery endothelial cells (HUAECs), atrium, vena cava, neutrophils, platelets, cerebral cortex, placenta, and samples from mesenchymal stem cell differentiation. In eight different samples from a single donor, we found highly tissue-specific circRNA expression. Circular-to-linear RNA ratios revealed that many circRNAs were expressed higher than their linear host transcripts. Among the 71 validated circRNAs, we noticed potential biomarkers. In adenosine deaminase-deficient, severe combined immunodeficiency (ADA-SCID) patients and in Wiskott-Aldrich-Syndrome (WAS) patients' samples, we found evidence for differential circRNA expression of genes that are involved in the molecular pathogenesis of both phenotypes. Our findings underscore the need to assess circRNAs in mechanisms of human disease. KEY MESSAGES: circRNA resource catalog of 20 clinically relevant tissues. circRNA expression is highly tissue-specific. circRNA transcripts are often more abundant than their linear host RNAs. circRNAs can be differentially expressed in disease-associated genes.

Clinical effects of phosphodiesterase 3A mutations in inherited hypertension with brachydactyly.

Autosomal-dominant hypertension with brachydactyly is a salt-independent Mendelian syndrome caused by activating mutations in the gene encoding phosphodiesterase 3A. These mutations increase the protein kinase A-mediated phosphorylation of phosphodiesterase 3A resulting in enhanced cAMP-hydrolytic affinity and accelerated cell proliferation. The phosphorylated vasodilator-stimulated phosphoprotein is diminished, and parathyroid hormone-related peptide is dysregulated, potentially accounting for all phenotypic features. Untreated patients die prematurely of stroke; however, hypertension-induced target-organ damage is otherwise hardly apparent. We conducted clinical studies of vascular function, cardiac functional imaging, platelet function in affected and nonaffected persons, and cell-based assays. Large-vessel and cardiac functions indeed seem to be preserved. The platelet studies showed normal platelet function. Cell-based studies demonstrated that available phosphodiesterase 3A inhibitors suppress the mutant isoforms. However, increasing cGMP to indirectly inhibit the enzyme seemed to have particular use. Our results shed more light on phosphodiesterase 3A activation and could be relevant to the treatment of severe hypertension in the general population.

PDE3A mutations cause autosomal dominant hypertension with brachydactyly.

Cardiovascular disease is the most common cause of death worldwide, and hypertension is the major risk factor. Mendelian hypertension elucidates mechanisms of blood pressure regulation. Here we report six missense mutations in PDE3A (encoding phosphodiesterase 3A) in six unrelated families with mendelian hypertension and brachydactyly type E (HTNB). The syndrome features brachydactyly type E (BDE), severe salt-independent but age-dependent hypertension, an increased fibroblast growth rate, neurovascular contact at the rostral-ventrolateral medulla, altered baroreflex blood pressure regulation and death from stroke before age 50 years when untreated. In vitro analyses of mesenchymal stem cell-derived vascular smooth muscle cells (VSMCs) and chondrocytes provided insights into molecular pathogenesis. The mutations increased protein kinase A-mediated PDE3A phosphorylation and resulted in gain of function, with increased cAMP-hydrolytic activity and enhanced cell proliferation. Levels of phosphorylated VASP were diminished, and PTHrP levels were dysregulated. We suggest that the identified PDE3A mutations cause the syndrome. VSMC-expressed PDE3A deserves scrutiny as a therapeutic target for the treatment of hypertension.