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.

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.

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. Key Words: arteriosclerosis Ⅲ endothelium Ⅲ immune system Ⅲ leukocytes E phrinB2 belongs to a group of membrane-associated ligands that bind to the largest family of receptor tyrosine kinases, the Eph receptors. 1 Genetic deficiency and loss-offunction mutations of ephrinB2 in mice lead to early embryonic lethality due to severe defects in arteriovenous differentiation. [2][3][4] Similarly, mice deficient in ephrinB2 receptors EphB2, EphB3, and EphB4 all display more or less severe vasculogenesis defects, with EphB4-deficient mice copying the phenotype of ephrinB2 knockout animals. 4 Although the ephrinB/EphB system has been established as an important determinant of vasculogenesis and angiogenesis, 5-7 it may also have a function in leukocyte activation. For instance, whereas EphB6-null mutations adversely affect T-cell function, 8,9 ephrinB2 can act as a costimulatory molecule for T cells inducing interferon-␥ release. 10 Moreover, functionally interfering with the ephrinB/EphB system impedes monocyte transmigration through an endothelial cell monolayer, 11,12 highlighting the potential pathophysiological relevance of ephrinB2-mediated endothelial cell-monocyte interactions. Conclusion-TheseHowever, whereas regulatory mechanisms have been characterized in some detail 4,13 -particularly vascular endothelial cell growth factor signaling controlling ephrinB2 expression in angiogenic endothelial cells-much less is known about ephrinB2 regulation and function in adult arteries. In this context, an increasing body of evidence suggests that endothelial cell ephrinB2 expression is affected by hemodynamic forces, such as laminar shear stress, which downregulates it in human endothelial cells. 14 In contrast, endothelial cells exposed t...

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.

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.

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.Subject Categories Vascular Biology & Angiogenesis

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.

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.