GPR124 regulates murine brain embryonic angiogenesis and BBB formation by an intracellular domain-independent mechanism.

The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical ß-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors.

Therapeutic blood-brain barrier modulation and stroke treatment by a bioengineered FZD4-selective WNT surrogate in mice.

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/ß-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

Regulation of the Blood-Brain Barrier in Health and Disease.

The neurovascular unit is a dynamic microenvironment with tightly controlled signaling and transport coordinated by the blood-brain barrier (BBB). A properly functioning BBB allows sufficient movement of ions and macromolecules to meet the high metabolic demand of the central nervous system (CNS), while protecting the brain from pathogenic and noxious insults. This review describes the main cell types comprising the BBB and unique molecular signatures of these cells. Additionally, major signaling pathways for BBB development and maintenance are highlighted. Finally, we describe the pathophysiology of BBB diseases, their relationship to barrier dysfunction, and identify avenues for therapeutic intervention.

Heat shock protein 27 activity is linked to endothelial barrier recovery after proinflammatory GPCR-induced disruption.

Vascular inflammation causes endothelial barrier disruption and tissue edema. Several inflammatory mediators act through G protein­coupled receptors (GPCRs), including protease-activated receptor-1 (PAR1), to elicit inflammatory responses. The activation of PAR1 by its ligand thrombin stimulates proinflammatory, p38 mitogen-activated protein kinase (MAPK) signaling that promotes endothelial barrier disruption. Through mass spectrometry phosphoproteomics, we identified heat shock protein 27 (HSP27), which exists as a large oligomer that binds to actin, as a promising candidate for the p38-mediated regulation of barrier integrity. Depletion of HSP27 by siRNA enhanced endothelial cell barrier permeability and slowed recovery after thrombin stimulation. We further showed that two effector kinases of p38 MAPK, MAPKAPK2 (MK2) and MAPKAPK3 (MK3), differentially phosphorylated HSP27 at Ser15, Ser78, and Ser82. Whereas inhibition of thrombin-stimulated p38 activation blocked HSP27 phosphorylation at all three sites, inhibition of MK2 reduced the phosphorylation of only Ser15 and Ser78. Inhibition of both MK2 and MK3 was necessary to attenuate Ser82 phosphorylation. Thrombin-stimulated p38-MK2-MK3 signaling induced HSP27 oligomer disassembly. However, a phosphorylation-deficient mutant of HSP27 exhibited defective oligomer disassembly and altered the dynamics of barrier recovery after thrombin stimulation. Moreover, blocking HSP27 oligomer reassembly with the small-molecule inhibitor J2 enhanced endothelial barrier permeability in vitro and vascular leakage in vivo in response to PAR1 activation. These studies reveal the distinct regulation of HSP27 phosphorylation and function induced by the GPCR-stimulated p38-MK2-MK3 signaling axis that controls the dynamics of endothelial barrier recovery in vitro and vascular leakage in vivo.

G protein-coupled receptors activate p38 MAPK via a non-canonical TAB1-TAB2- and TAB1-TAB3-dependent pathway in endothelial cells.

Endothelial dysfunction is induced by inflammatory mediators including multiple G protein-coupled receptor (GPCR) agonists. However, the GPCR signaling pathways that promote endothelial dysfunction are incompletely understood. We previously showed that thrombin promotes endothelial barrier disruption through autophosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) via a non-canonical transforming growth factor-ß-activated protein kinase-1-binding protein-1 (TAB1) and TAB2-dependent pathway rather than the canonical three-tiered kinase cascade. Here, we sought to determine whether other GPCR agonists stimulate p38 MAPK activation via this non-canonical pathway in human endothelial cells derived from different vascular beds. Using primary human umbilical vein endothelial cells (HUVECs), HUVEC-derived EA.hy926 cells, and human dermal microvascular endothelial cells (HDMECs), we found that both non-canonical and canonical p38 activation pathways components are expressed in these various endothelial cell types, including TAB3, a structurally-related TAB2 homolog. Moreover, multiple GPCRs agonists, including thrombin, histamine, prostaglandin E2, and ADP, stimulated robust p38 autophosphorylation, whereas phosphorylation of the upstream MAPKs MAP kinase kinase 3 (MKK3) and MKK6, was virtually undetectable, indicating that non-canonical p38 activation may exist for other GPCRs. Indeed, in EA.hy926 cells, thrombin- and histamine-stimulated p38 activation depended on TAB1-TAB2, whereas in primary HUVECs, both TAB1-TAB2 and TAB1-TAB3 were required for p38 activation. In HDMECs, thrombin-induced p38 activation depended on TAB1-TAB3, but histamine-induced p38 activation required TAB1-TAB2. Moreover, thrombin- and histamine-stimulated interleukin-6 production required both TAB1-TAB2 and TAB1-TAB3 in HUVEC. We conclude that multiple GPCR agonists utilize non-canonical TAB1-TAB2 and TAB1-TAB3-dependent p38 activation to promote endothelial inflammatory responses.

A Tyrosine Switch on NEDD4-2 E3 Ligase Transmits GPCR Inflammatory Signaling.

Ubiquitination is essential for protein degradation and signaling and pivotal to many physiological processes. Ubiquitination of a subset of G-protein-coupled receptors (GPCRs) by the E3 ligase NEDD4-2 is required for p38 activation, but how GPCRs activate NEDD4-2 to promote ubiquitin-mediated signaling is not known. Here, we report that the GPCR protease-activated receptor-1 (PAR1) stimulates c-Src-mediated tyrosine phosphorylation and activation of NEDD4-2 to promote p38 signaling and endothelial barrier disruption. Using mass spectrometry, we identified a unique phosphorylated tyrosine (Y)-485 within the 2,3-linker peptide between WW domain 2 and 3 of NEDD4-2 in agonist-stimulated cells. Mutation of NEDD4-2 Y485 impaired E3 ligase activity and failed to rescue PAR1-stimulated p38 activation and endothelial barrier permeability. The purinergic P2Y1 receptor also required c-Src and NEDD4-2 tyrosine phosphorylation for p38 activation. These studies reveal a novel role for c-Src in GPCR-induced NEDD4-2 activation, which is critical for driving ubiquitin-mediated p38 inflammatory signaling.

Intrauterine telemetry to measure mouse contractile pressure in vivo.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

The SK3 channel promotes placental vascularization by enhancing secretion of angiogenic factors.

Proper placental perfusion is essential for fetal exchange of oxygen, nutrients, and waste with the maternal circulation. Impairment of uteroplacental vascular function can lead to pregnancy complications, including preeclampsia and intrauterine growth restriction (IUGR). Potassium channels have been recognized as regulators of vascular proliferation, angiogenesis, and secretion of vasoactive factors, and their dysfunction may underlie pregnancy-related vascular diseases. Overexpression of one channel in particular, the small-conductance calcium-activated potassium channel 3 (SK3), is known to increase vascularization in mice, and mice overexpressing the SK3 channel (SK3(T/T) mice) have a high rate of fetal demise and IUGR. Here, we show that overexpression of SK3 causes fetal loss through abnormal placental vascularization. We previously reported that, at pregnancy day 14, placentas isolated from SK3(T/T) mice are smaller than those obtained from wild-type mice. In this study, histological analysis reveals that SK3(T/-) placentas at this stage have abnormal placental morphology, and microcomputed tomography shows that these placentas have significantly larger and more blood vessels than those from wild-type mice. To identify the mechanism by which these vascularization defects occur, we measured levels of vascular endothelial growth factor (VEGF), placental growth factor, and the soluble form of VEGF receptor 1 (sFlt-1), which must be tightly regulated to ensure proper placental development. Our data reveal that overexpression of SK3 alters systemic and placental ratios of the angiogenic factor VEGF to antiangiogenic factor sFlt-1 throughout pregnancy. Additionally, we observe increased expression of hypoxia-inducing factor 2α in SK3(T/-) placentas. We conclude that the SK3 channel modulates placental vascular development and fetal health by altering VEGF signaling.

The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy.

Abnormal uterine activity in pregnancy causes a range of important clinical disorders, including preterm birth, dysfunctional labour and post-partum haemorrhage. Uterine contractile patterns are controlled by the generation of complex electrical signals at the myometrial smooth muscle plasma membrane. To identify novel targets to treat conditions associated with uterine dysfunction, we undertook a genome-wide screen of potassium channels that are enriched in myometrial smooth muscle. Computational modelling identified Kir7.1 as potentially important in regulating uterine excitability during pregnancy. We demonstrate Kir7.1 current hyper-polarizes uterine myocytes and promotes quiescence during gestation. Labour is associated with a decline, but not loss, of Kir7.1 expression. Knockdown of Kir7.1 by lentiviral expression of miRNA was sufficient to increase uterine contractile force and duration significantly. Conversely, overexpression of Kir7.1 inhibited uterine contractility. Finally, we demonstrate that the Kir7.1 inhibitor VU590 as well as novel derivative compounds induces profound, long-lasting contractions in mouse and human myometrium; the activity of these inhibitors exceeds that of other uterotonic drugs. We conclude Kir7.1 regulates the transition from quiescence to contractions in the pregnant uterus and may be a target for therapies to control uterine contractility.

Overexpression of the SK3 channel alters vascular remodeling during pregnancy, leading to fetal demise.

The maternal cardiovascular system undergoes hemodynamic changes during pregnancy via angiogenesis and vasodilation to ensure adequate perfusion of the placenta. Improper vascularization at the maternal-fetal interface can cause pregnancy complications and poor fetal outcomes. Recent evidence indicates that small conductance Ca(2+)-activated K(+) channel subtype 3 (SK3) contributes to vascular remodeling during pregnancy, and we hypothesized that abnormal SK3 channel expression would alter the ability of the maternal cardiovascular system to adapt to pregnancy demands and lead to poor fetal outcomes. We investigated this hypothesis using transgenic Kcnn3(tm1Jpad)/Kcnn3(tm1Jpad) (SK3(T/T)) mice that overexpress the channel. Isolated pressurized uterine arteries from nonpregnant transgenic SK3(T/T) mice had larger basal diameters and decreased agonist-induced constriction than those from their wild-type counterparts; however, non-receptor-mediated depolarization remained intact. In addition to vascular changes, heart rates and ejection fraction were increased, whereas end systolic volume was reduced in SK3(T/T) mice compared with their wild-type littermates. Uterine sonography of the fetuses on pregnancy day 14 showed a significant decrease in fetal size in SK3(T/T) compared with wild-type mice; thus, SK3(T/T) mice displayed an intrauterine growth-restricted phenotype. The SK3(T/T) mice showed decreased placental thicknesses and higher incidence of fetal loss, losing over half of their complement of pups by midgestation. These results establish that the SK3 channel contributes to both maternal and fetal outcomes during pregnancy and point to the importance of SK3 channel regulation in maintaining a healthy pregnancy.

Altered contribution of RhoA/Rho kinase signaling in contractile activity of myometrium in leptin receptor-deficient mice.

In late gestation, enhanced myometrial contractility is mediated in part through increased Rho/Rho kinase. Since leptin, which is elevated in pregnancy and obesity, can directly depress myometrial function, we hypothesized that in leptin receptor-deficient mice, myometrial contractility would be greater in late pregnancy due to increased Rho/Rho kinase activity. To test this, we correlated RhoA and Rho kinase expression to contractility in myometrium from nonpregnant (NP) and late-pregnant (P18) heterozygous leptin receptor-deficient mice (db/+) vs. wild-type (WT) mice. In NP mice, KCl-induced contractions were similar between WT and db/+ myometrium. However, the Rho kinase-dependent component of the contractions was greater in db/+ mice, along with an increased expression of Rho kinase. KCl-induced contractions increased in strength in myometrium from P18 WT and db/+ compared with NP. Although the contribution of Rho kinase to contractions was unchanged in P18 WT mice, it was decreased in P18 db/+ mice. The decrease in Rho kinase-dependent contractions in P18 db/+ mice coincided with reduced RhoA and Rho kinase expression relative to NP db/+. Addition of high-fat-induced abnormal glucose utilization prevented changes in Rho kinase function. We conclude that abnormal leptin signaling increases expression and function of Rho kinase to maintain contractile function in NP myometrium and that during pregnancy the contribution of RhoA and Rho kinase expression to myometrial function is reduced despite an increase in myometrial contractility. Thus, other signaling mechanisms appear to compensate when leptin signaling is reduced to maintain contractile function during pregnancy.