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Supplemental Digital Content is available in the text., Background: PALMD (palmdelphin) belongs to the family of paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis and predict severity of the disease. Methods: Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, coimmunoprecipitation, and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in small interferring RNA-treated EC cultures, knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the single nucleotide polymorphism rs7543130 informed about gene regulatory changes. Results: ECs express the cytosolic PALMD-KKVI splice variant, which associated with RANGAP1 (RAN GTP hydrolyase activating protein 1). RANGAP1 regulates the activity of the GTPase RAN and thereby nucleocytoplasmic shuttling via XPO1 (Exportin1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently in gene regulation because of the effect on localization of transcriptional regulators. Changes in EC responsiveness on loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd−/− mouse aorta, and human aortic valve samples derived from patients with calcific aortic valve stenosis. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs. Conclusions: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD’s contribution to calcific aortic valve stenosis pathology.

Aim Protein kinase (PK) A anchoring protein (AKAP) 12 is a scaffolding protein that anchors PKA to compartmentalize cyclic AMP signalling. This study assessed the consequences of the downregulation or deletion of AKAP12 on endothelial cell migration and angiogenesis. Methods The consequences of siRNA‐mediated downregulation AKAP12 were studied in primary cultures of human endothelial cells as well as in endothelial cells and retinas from wild‐type versus AKAP12−/− mice. Molecular interactions were investigated using a combination of immunoprecipitation and mass spectrometry. Results AKAP12 was expressed at low levels in confluent endothelial cells but its expression was increased in actively migrating cells, where it localized to lamellipodia. In the postnatal retina, AKAP12 was expressed by actively migrating tip cells at the angiogenic front, and its deletion resulted in defective extension of the vascular plexus. In migrating endothelial cells, AKAP12 was co‐localized with the PKA type II‐α regulatory subunit as well as multiple key regulators of actin dynamics and actin filament‐based movement; including components of the Arp2/3 complex and the vasodilator‐stimulated phosphoprotein (VASP). Fitting with the evidence of a physical VASP/AKAP12/PKA complex, it was possible to demonstrate that the VEGF‐stimulated and PKA‐dependent phosphorylation of VASP was dependent on AKAP12. Indeed, AKAP12 colocalized with phospho‐Ser157 VASP at the leading edge of migrating endothelial cells. Conclusion The results suggest that compartmentalized AKAP12/PKA signalling mediates VASP phosphorylation at the leading edge of migrating endothelial cells to translate angiogenic stimuli into altered actin dynamics and cell movement.

Angiogenesis, an integral part of many physiological and pathological processes, is a tightly regulated multistep process. Angiogenesis assays are used to clarify the molecular mechanisms and screen for pharmacological inhibitors. However, most in vitro angiogenesis models measure only one aspect of this process, whereas in vivo assays are complex and difficult to interpret. The ex vivo aortic ring model allows the study of many key features of angiogenesis, such as endothelial activation, branching, and remodeling as well as later steps such as pericyte acquisition. This model can be modified to include genetic manipulation and can be used to assess the pro- or anti-angiogenic effects of compounds in a relatively controlled system.