Anja Feldner

Synaptojanin-2 binding protein stabilizes the Notch ligands DLL1 and DLL4 and inhibits sprouting angiogenesis.

Rationale: The formation of novel blood vessels is initiated by vascular endothelial growth factor. Subsequently, DLL4-Notch signaling controls the selection of tip cells, which guide new sprouts, and trailing stalk cells. Notch signaling in stalk cells is induced by DLL4 on the tip cells. Moreover, DLL4 and DLL1 are expressed in the stalk cell plexus to maintain Notch signaling. Notch loss-of-function causes formation of a hyperdense vascular network with disturbed blood flow. Objective: This study was aimed at identifying novel modifiers of Notch signaling that interact with the intracellular domains of DLL1 and DLL4. Methods and Results: Synaptojanin-2 binding protein (SYNJ2BP, also known as ARIP2) interacted with the PDZ binding motif of DLL1 and DLL4, but not with the Notch ligand Jagged-1. SYNJ2BP was preferentially expressed in stalk cells, enhanced DLL1 and DLL4 protein stability, and promoted Notch signaling in endothelial cells. SYNJ2BP induced expression of the Notch target genes HEY1, lunatic fringe (LFNG), and ephrin-B2, reduced phosphorylation of ERK1/2, and decreased expression of the angiogenic factor vascular endothelial growth factor (VEGF)-C. It inhibited the expression of genes enriched in tip cells, such as angiopoietin-2, ESM1, and Apelin, and impaired tip cell formation. SYNJ2BP inhibited endothelial cell migration, proliferation, and VEGF-induced angiogenesis. This could be rescued by blockade of Notch signaling or application of angiopoietin-2. SYNJ2BP-silenced human endothelial cells formed a functional vascular network in immunocompromised mice with significantly increased vascular density. Conclusions: These data identify SYNJ2BP as a novel inhibitor of tip cell formation, executing its functions predominately by promoting Delta-Notch signaling.

Hypertension impairs myocardin function: a novel mechanism facilitating arterial remodelling

AIMS Hypertension evokes detrimental changes in the arterial vessel wall that facilitate stiffening and thus lead to a further rise in mean blood pressure, eventually causing heart failure. The underlying pathophysiological remodelling process is elicited by an increase in wall stress (WS) and is strictly dependent on the activation of vascular smooth muscle cells (SMC). However, it remains unclear as to why these cells fail to maintain their contractile and quiescent phenotype in a hypertensive environment. METHODS AND RESULTS In this context, we reveal that the knockdown of myocardin--a pivotal transcriptional determinant of the contractile SMC phenotype--is sufficient to induce SMC proliferation. In line with this observation, immunofluorescence analysis of the media of remodelling arteries from hypertensive mice demonstrated a significant decrease in the abundance of myocardin and an increase in SMC proliferation. Subsequent analyses of isolated perfused mouse arteries and human cultured SMCs exposed to cyclic stretch (i.e. mimicking one component of WS) suggested that this biomechanical force facilitates serine phosphorylation of myocardin. Furthermore, this biomechanical stimulus promotes rapid translocation of myocardin from the nucleus to the cytoplasm, inhibits its mRNA expression, and causes proteasomal degradation of the cytoplasmic protein. CONCLUSIONS Collectively, these findings suggest that hypertension negates the activity of myocardin in SMCs on multiple levels, hence eliminating a crucial determinant of SMC quiescence. This mechanism may control the initial switch from the contractile towards the synthetic SMC phenotype during hypertension and may offer an interesting novel approach to prevent cardiovascular disease.

Endothelial Cell EphrinB2-Dependent Activation of Monocytes in Arteriosclerosis

Objective—The expression of ephrinB2 in endothelial cells delineates their arterial phenotype and is a prerequisite for the development of the embryonic vasculature. Whereas the role of ephrinB2 in the microcirculation has been studied extensively, its expression and function in adult arteries is hardly understood. Methods and Results—Our analyses showed that in mouse arteries, ephrinB2 is located on the luminal surface of endothelial cells and may physically interact with monocyte EphB receptors. Moreover, transdifferentiation of human monocytes into macrophages was associated with an increase in EphB2 expression, and exposing monocytes to immobilized ephrinB2 resulted in phosphorylation of the receptor followed by an increased expression of proinflammatory chemokines such as interleukin-8 and monocyte chemotactic protein-1/CCL2. Relating to the (patho)physiological relevance of these findings, immunofluorescence analyses revealed that ephrinB2 is most abundantly expressed in endothelial cells at arteriosclerosis predilection sites of the mouse aorta. Subsequent analyses indicated that monocyte adhesion to aortic segments abundantly expressing ephrinB2 is strongly enhanced and that endothelial cell ephrinB2 forward signaling is sufficient to upregulate cytokine expression in monocytes. Conclusion—These observations suggest a hitherto unknown link between vascular ephrinB2 expression and the proinflammatory activation of monocytes that may contribute to the pathogenesis of arteriosclerosis.

Junb regulates arterial contraction capacity, cellular contractility, and motility via its target Myl9 in mice

Cellular contractility and, thus, the ability to alter cell shape are prerequisites for a number of important biological processes such as cytokinesis, movement, differentiation, and substrate adherence. The contractile capacity of vascular smooth muscle cells (VSMCs) is pivotal for the regulation of vascular tone and thus blood pressure and flow. Here, we report that conditional ablation of the transcriptional regulator Junb results in impaired arterial contractility in vivo and in vitro. This was exemplified by resistance of Junb-deficient mice to DOCA-salt-induced volume-dependent hypertension as well as by a decreased contractile capacity of isolated arteries. Detailed analyses of Junb-deficient VSMCs, mouse embryonic fibroblasts, and endothelial cells revealed a general failure in stress fiber formation and impaired cellular motility. Concomitantly, we identified myosin regulatory light chain 9 (Myl9), which is critically involved in actomyosin contractility and stress fiber assembly, as a Junb target. Consistent with these findings, reexpression of either Junb or Myl9 in Junb-deficient cells restored stress fiber formation, cellular motility, and contractile capacity. Our data establish a molecular link between the activator protein-1 transcription factor subunit Junb and actomyosin-based cellular motility as well as cellular and vascular contractility by governing Myl9 transcription.

RGS5 promotes arterial growth during arteriogenesis.

Arteriogenesis—the growth of collateral arterioles—partially compensates for the progressive occlusion of large conductance arteries as it may occur as a consequence of coronary, cerebral or peripheral artery disease. Despite being clinically highly relevant, mechanisms driving this process remain elusive. In this context, our study revealed that abundance of regulator of G‐protein signalling 5 (RGS5) is increased in vascular smooth muscle cells (SMCs) of remodelling collateral arterioles. RGS5 terminates G‐protein‐coupled signalling cascades which control contractile responses of SMCs. Consequently, overexpression of RGS5 blunted Gαq/11‐mediated mobilization of intracellular calcium, thereby facilitating Gα12/13‐mediated RhoA signalling which is crucial for arteriogenesis. Knockdown of RGS5 evoked opposite effects and thus strongly impaired collateral growth as evidenced by a blockade of RhoA activation, SMC proliferation and the inability of these cells to acquire an activated phenotype in RGS5‐deficient mice after the onset of arteriogenesis. Collectively, these findings establish RGS5 as a novel determinant of arteriogenesis which shifts G‐protein signalling from Gαq/11‐mediated calcium‐dependent contraction towards Gα12/13‐mediated Rho kinase‐dependent SMC activation.

Experimental hypertension triggers varicosis-like maladaptive venous remodeling through activator protein-1

An increase in circumferential wall tension (CWT) is an important determinant of vascular remodeling during hypertension or arteriosclerosis but also arteriogenesis. Although pivotal for such processes, the effect of this biomechanical force on venous remodeling has not yet been delineated. To this end, we raised the filling pressure in veins of the mouse auricle, which led to a 2.5‐fold enlargement of these blood vessels within 4 d along with an increase in smooth muscle cell proliferation, matrix metalloproteinase 2 (MMP‐2) expression and gelatinase activity. These changes were likewise observed in tissue samples of human varicose veins. Topical treatment of the auricles with a decoy oligonucleotide‐neutralizing activator protein 1 (AP‐1) inhibited these effects. Likewise, proliferation, MMP‐2 expression, and gelatinase activity in both native and cultured venous smooth muscle cells exposed to enhanced stretch was decreased by up to 80% through inhibiting AP‐1. In contrast, mutant control oligonucleotides had no effect on smooth muscle cell activation. These findings indicate that an increase in venous filling pressure and thus CWT is sufficient to activate AP‐1, which, in turn, triggers varicose remodeling through fuelling MMP‐2 activity and smooth muscle cell hyperplasia in the venous vessel wall.—Feldner, A., Otto, H., Rewerk, S., Hecker, M., Korff, T. Experimental hypertension triggers varicosis‐like maladaptive venous remodeling through activator protein‐1. FASEB J. 25, 3613–3621 (2011). www.fasebj.org

Angiopoietin-1 mediates inhibition of hypertension-induced release of angiopoietin-2 from endothelial cells

AIMS Adequate endothelial cell stimulation is a prerequisite for the adaptive remodelling of macro- and microvessels. A pivotal autocrine mechanism following endothelial cell activation is the release of angiopoietin-2 (Ang-2), which subsequently antagonizes the binding of Ang-1 to the Tie-2 receptor, thus sensitizing the endothelial cells to pro-angiogenic and/or pro-inflammatory stimuli. Based on the observation that hypertension in mice reduces the abundance of Ang-2 stored in arterial endothelial cells, this study was aimed at testing the hypothesis that an increase in wall stress (WS) or stretch-a hallmark of hypertension-is sufficient to release Ang-2 from endothelial cells. METHODS AND RESULTS In fact, stretching of isolated perfused mouse arteries or human cultured endothelial cells rapidly elicited an increased release of Ang-2. In the cultured endothelial cells, this was preceded by a transient rise in intracellular free calcium, abrogated through calcium chelation and accompanied by a decrease in Tie-2 phosphorylation. Interestingly, Ang-1 abolished the stretch-induced release of Ang-2 from both cultured and native endothelial cells through inhibiting the stretch-dependent mobilization of intracellular calcium. CONCLUSION Collectively, these results indicate that increased WS or stretch facilitates the release of Ang-2 from endothelial cell Weibel-Palade bodies, and that Ang-1 can block this by attenuating the stretch-mediated rise in intracellular calcium.

No Evidence for a Functional Role of Bi-Directional Notch Signaling during Angiogenesis

The Delta-Notch pathway is a signal exchanger between adjacent cells to regulate numerous differentiation steps during embryonic development. Blood vessel formation by sprouting angiogenesis requires high expression of the Notch ligand DLL4 in the leading tip cell, while Notch receptors in the trailing stalk cells are activated by DLL4 to achieve strong Notch signaling activity. Upon ligand binding, Notch receptors are cleaved by ADAM proteases and gamma-secretase. This releases the intracellular Notch domain that acts as a transcription factor. There is evidence that also Notch ligands (DLL1, DLL4, JAG1, JAG2) are processed upon receptor binding to influence transcription in the ligand-expressing cell. Thus, the existence of bi-directional Delta-Notch signaling has been proposed. We report here that the Notch ligands DLL1 and JAG1 are processed in endothelial cells in a gamma-secretase-dependent manner and that the intracellular ligand domains accumulate in the cell nucleus. Overexpression of JAG1 intracellular domain (ICD) as well as DLL1-ICD, DLL4-ICD and NOTCH1-ICD inhibited endothelial proliferation. Whereas NOTCH1-ICD strongly repressed endothelial migration and sprouting angiogenesis, JAG1-ICD, DLL1-ICD and DLL4-ICD had no significant effects. Consistently, global gene expression patterns were only marginally affected by the processed Notch ligands. In addition to its effects as a transcription factor, NOTCH1-ICD promotes cell adhesion to the extracellular matrix in a transcription-independent manner. However, JAG1-ICD, DLL1-ICD and DLL4-ICD did not influence endothelial cell adhesion. In summary, reverse signaling of Notch ligands appears to be dispensable for angiogenesis in cellular systems.

Bortezomib protects from varicose-like venous remodeling

Despite the high prevalence of venous diseases that are associated with and based on the structural reorganization of the venous vessel wall, not much is known about their mechanistic causes. In this context, we demonstrated that the quantity of myocardin, a transcriptional regulator of the contractile and quiescent smooth muscle cell phenotype, was diminished in proliferating synthetic venous smooth muscle cells (VSMCs) of human and mouse varicose veins by 51 and 60%, respectively. On the basis of the relevance of proteasomal activity for such phenotypic changes, we hypothesized that the observed VSMC activation is attenuated by the proteasome inhibitor bortezomib. This drug fully abolished VSMC proliferation and loss of myocardin in perfused mouse veins and blocked VSMC invasion in collagen gels by almost 80%. In line with this, topical transdermal treatment with bortezomib diminished VSMC proliferation by 80%, rescued 90% of VSMC myocardin abundance, and inhibited varicose‐like venous remodeling by 67 to 72% in a mouse model. Collectively, our data indicate that the proteasome plays a pivotal role in VSMC phenotype changes during venous remodeling processes. Its inhibition protects from varicose‐like vein remodeling in mice and may thus serve as a putative therapeutic strategy to treat human varicose veins.—Pfisterer, L., Meyer, R., Feldner, A., Drews, O., Hecker, M., Korff, T. Bortezomib protects from varicose‐like venous remodeling. FASEB J. 28, 3518–3527 (2014). www.fasebj.org

Loss of Mpdz impairs ependymal cell integrity leading to perinatal‐onset hydrocephalus in mice

Hydrocephalus is a common congenital anomaly. LCAM1 and MPDZ (MUPP1) are the only known human gene loci associated with non‐syndromic hydrocephalus. To investigate functions of the tight junction‐associated protein Mpdz, we generated mouse models. Global Mpdz gene deletion or conditional inactivation in Nestin‐positive cells led to formation of supratentorial hydrocephalus in the early postnatal period. Blood vessels, epithelial cells of the choroid plexus, and cilia on ependymal cells, which line the ventricular system, remained morphologically intact in Mpdz‐deficient brains. However, flow of cerebrospinal fluid through the cerebral aqueduct was blocked from postnatal day 3 onward. Silencing of Mpdz expression in cultured epithelial cells impaired barrier integrity, and loss of Mpdz in astrocytes increased RhoA activity. In Mpdz‐deficient mice, ependymal cells had morphologically normal tight junctions, but expression of the interacting planar cell polarity protein Pals1 was diminished and barrier integrity got progressively lost. Ependymal denudation was accompanied by reactive astrogliosis leading to aqueductal stenosis. This work provides a relevant hydrocephalus mouse model and demonstrates that Mpdz is essential to maintain integrity of the ependyma.