Phospholipase Cε hydrolyzes perinuclear phosphatidylinositol 4-phosphate to regulate cardiac hypertrophy.

Phospholipase Cε (PLCε) is a multifunctional enzyme implicated in cardiovascular, pancreatic, and inflammatory functions. Here we show that conditional deletion of PLCε in mouse cardiac myocytes protects from stress-induced pathological hypertrophy. PLCε small interfering RNA (siRNA) in ventricular myocytes decreases endothelin-1 (ET-1)-dependent elevation of nuclear calcium and activation of nuclear protein kinase D (PKD). PLCε scaffolded to muscle-specific A kinase-anchoring protein (mAKAP), along with PKCε and PKD, localizes these components at or near the nuclear envelope, and this complex is required for nuclear PKD activation. Phosphatidylinositol 4-phosphate (PI4P) is identified as a perinuclear substrate in the Golgi apparatus for mAKAP-scaffolded PLCε. We conclude that perinuclear PLCε, scaffolded to mAKAP in cardiac myocytes, responds to hypertrophic stimuli to generate diacylglycerol (DAG) from PI4P in the Golgi apparatus, in close proximity to the nuclear envelope, to regulate activation of nuclear PKD and hypertrophic signaling pathways.

Endothelial PHACTR1 Promotes Endothelial Activation and Atherosclerosis by Repressing PPARγ Activity Under Disturbed Flow in Mice.

BACKGROUND: Numerous genome-wide association studies revealed that SNPs (single nucleotide polymorphisms) at the PHACTR1 (phosphatase and actin regulator 1) locus strongly correlate with coronary artery disease. However, the biological function of PHACTR1 remains poorly understood. Here, we identified the proatherosclerotic effect of endothelial PHACTR1, contrary to macrophage PHACTR1. METHODS: We generated global (Phactr1-/-) and endothelial cell (EC)-specific (Phactr1ECKO) Phactr1 KO (knockout) mice and crossed these mice with apolipoprotein E-deficient (Apoe-/-) mice. Atherosclerosis was induced by feeding the high-fat/high-cholesterol diet for 12 weeks or partially ligating carotid arteries combined with a 2-week high-fat/high-cholesterol diet. PHACTR1 localization was identified by immunostaining of overexpressed PHACTR1 in human umbilical vein ECs exposed to different types of flow. The molecular function of endothelial PHACTR1 was explored by RNA sequencing using EC-enriched mRNA from global or EC-specific Phactr1 KO mice. Endothelial activation was evaluated in human umbilical vein ECs transfected with siRNA targeting PHACTR1 and in Phactr1ECKO mice after partial carotid ligation. RESULTS: Global or EC-specific Phactr1 deficiency significantly inhibited atherosclerosis in regions of disturbed flow. PHACTR1 was enriched in ECs and located in the nucleus of disturbed flow areas but shuttled to cytoplasm under laminar flow in vitro. RNA sequencing showed that endothelial Phactr1 depletion affected vascular function, and PPARγ (peroxisome proliferator-activated receptor gamma) was the top transcription factor regulating differentially expressed genes. PHACTR1 functioned as a PPARγ transcriptional corepressor by binding to PPARγ through the corepressor motifs. PPARγ activation protects against atherosclerosis by inhibiting endothelial activation. Consistently, PHACTR1 deficiency remarkably reduced endothelial activation induced by disturbed flow in vivo and in vitro. PPARγ antagonist GW9662 abolished the protective effects of Phactr1 KO on EC activation and atherosclerosis in vivo. CONCLUSIONS: Our results identified endothelial PHACTR1 as a novel PPARγ corepressor to promote atherosclerosis in disturbed flow regions. Endothelial PHACTR1 is a potential therapeutic target for atherosclerosis treatment.

Vessel tech: a high-accuracy pipeline for comprehensive mouse retinal vasculature characterization.

Mouse retinal vasculature is a well-recognized and commonly used animal model for angiogenesis and microvascular remodeling. Morphological features of retinal vasculature reflect the vessel's biological functions, and are critical in understanding the physiological and pathological process of vascular development and disease. Here we developed a comprehensive software, Vessel Tech, using retinal vasculature images of postnatal mice. This pipeline can automatically process retinal vascular images, reconstruct vessel network with high accuracy and assess global and local vascular characteristics based on the recent machine-learning techniques. The development of Vessel Tech provides a powerful tool for vascular biologists.

Rare Pulmonary Connective Tissue Type Mast Cells Regulate Lung Endothelial Cell Angiogenesis.

Within the human lung, mast cells typically reside adjacent to the conducting airway and assume a mucosal phenotype (MCT). In rare pathologic conditions, connective tissue phenotype mast cells (MCTCs) can be found in the lung parenchyma. MCTCs accumulate in the lungs of infants with severe bronchopulmonary dysplasia, a chronic lung disease associated with preterm birth, which is characterized by pulmonary vascular dysmorphia. The human mast cell line (LUVA) was used to model MCTCs or MCTs. The ability of MCTCs to affect vascular organization during fetal lung development was tested in mouse lung explant cultures. The effect of MCTCs on in vitro tube formation and barrier function was studied using primary fetal human pulmonary microvascular endothelial cells. The mechanistic role of MCTC proteases was tested using inhibitors. MCTCLUVA but not MCTLUVA was associated with vascular dysmorphia in lung explants. In vitro, the addition of MCTCLUVA potentiated fetal human pulmonary microvascular endothelial cell interactions, inhibited tube stability, and disrupted endothelial cell junctions. Protease inhibitors ameliorated the ability of MCTCLUVA to alter endothelial cell angiogenic activities in vitro and ex vivo. These data indicate that MCTCs may directly contribute to disrupted angiogenesis in bronchopulmonary dysplasia. A better understanding of factors that regulate mast cell subtype and their different effector functions is essential.

Dll4-Notch1 signaling but not VEGF-A is essential for hyperoxia induced vessel regression in retina.

It is well recognized that decreased vascular endothelial growth factor A (VEGF-A) mRNA plays an important role in retinal vessel regression induced by hyperoxia. However, this concept has been challenged by increasing new evidence. Furthermore, VEGF-A strongly enhances Dll4 expression and inhibition of Dll4-Notch signaling leads to excessive sprouting angiogenesis. Recently, it is shown that inactivation of Dll4-Notch1 signaling reduce hyperoxia induced vessel regression. It is unknown whether sprouting angiogenesis contributes to the protective effect or not and further investigations are needed. Moreover, the expression of Dll4 or Notch1 activation in the regressing plexus remains elucidated. To determine the role of VEGF-A and Dll4-Notch1 signaling in hyperoxia induced vascular regression in the retina, we used mice at postnatal day 5 (P5) - P7. Hyperoxia induced massive vascular regression in the central plexus but not in the angiogenic plexus and had no effect on sprouting angiogenesis. Immunostaining showed that VEGF-A was significantly repressed in the angiogenic front region after hyperoxia exposure but not detectable in the central area of both normoxia and hyperoxia treated retinas. In contrast, Notch ligand Delta-like 4 (Dll4) and Notch1 intracellular domain (N1-ICD) expression were inhibited in the regressing capillaries of central retina but comparable in the angiogenic plexus after high oxygen treatment. Moreover, administration of Dll4 neutralizing antibody or γ-Secretase inhibitor DAPT significantly aggravated vessel regression induced by short-time hyperoxia administration. Our data show that repressed Dll4-Notch1 signaling pathway but not downregulation of VEGF-A expression are responsible for hyperoxia induced pervasive vessel regression.

XAV939 Inhibits Intima Formation by Decreasing Vascular Smooth Muscle Cell Proliferation and Migration Through Blocking Wnt Signaling.

BACKGROUND: Excessive proliferation, migration, and oxidative stress of vascular smooth muscle cells (VSMCs) are key mechanisms involved in intima formation, which is the basic pathological process of in stent restenosis. This study aims at exploring the role of XAV939 in proliferation, migration, and reactive oxygen species (ROS) generation of VSMCs, and hence evaluating its effects on intima formation. METHODS: Carotid artery ligation models for C57BL/6 mice were established and gave them different intervention: saline, XAV939, Axin2 overexpression adenovirus, and negative control adenovirus. The intima formation was assayed by intima area and intima/media ratio. To investigate the underlying mechanisms, primary rat VSMCs were cultured and treated with XAV939 and platelet-derived growth factor-BB. EdU, direct cell counting, cell wound-healing assay, and flow cytometry were used to measure proliferation, migration, cell cycle, apoptosis, and ROS generation of VSMCs, respectively. By Western blot, we examined proliferating cell nuclear antigen, Cyclin D1, Cyclin E, p21, ß-actin, JNK, phosphorylated JNK, Axin2 and ß-catenin expression. Immunofluorescence staining and confocal microscopy were conducted to detect translocation of ß-catenin. RESULTS: XAV939 inhibited intima formation, which was exhibited by the loss of intima area and I/M ratio and attenuated proliferation, migration, and ROS generation, as well as promoted cell cycle arrest of VSMCs. Specifically, XAV939 inhibited Wnt pathway. CONCLUSIONS: XAV939 attenuates intima formation because of its inhibition of proliferation, migration, and apoptosis of VSMCs through suppression of Wnt signaling pathway.

G-protein-coupled receptor-2-interacting protein-1 is required for endothelial cell directional migration and tumor angiogenesis via cortactin-dependent lamellipodia formation.

OBJECTIVE: Recent evidence suggests G-protein-coupled receptor-2-interacting protein-1 (GIT1) overexpression in several human metastatic tumors, including breast, lung, and prostate. Tumor metastasis is associated with an increase in angiogenesis. We have showed previously that GIT1 is required for postnatal angiogenesis during lung development. However, the functional role of GIT1 in pathological angiogenesis during tumor growth is unknown. APPROACH AND RESULTS: In the present study, we show inhibition of angiogenesis in matrigel implants as well as reduced tumor angiogenesis and melanoma tumor growth in GIT1-knockout mice. We demonstrate that this is a result of impaired directional migration of GIT1-depleted endothelial cells toward a vascular endothelial growth factor gradient. Cortactin-mediated lamellipodia formation in the leading edge is critical for directional migration. We observed a significant reduction in cortactin localization and lamellipodia formation in the leading edge of GIT1-depleted endothelial cells. We specifically identified that the Spa homology domain (aa 250-420) of GIT1 is required for GIT1-cortactin complex localization to the leading edge. The mechanisms involved extracellular signal-regulated kinases 1 and 2-mediated Cortactin-S405 phosphorylation and activation of Rac1/Cdc42. Finally, using gain of function studies, we show that a constitutively active mutant of cortactin restored directional migration of GIT1-depleted cells. CONCLUSION: Our data demonstrated that a GIT1-cortactin association through GIT1-Spa homology domain is required for cortactin localization to the leading edge and is essential for endothelial cell directional migration and tumor angiogenesis.

Thioredoxin-interacting protein mediates sustained VEGFR2 signaling in endothelial cells required for angiogenesis.

OBJECTIVE: Thioredoxin-interacting protein (TXNIP) is an α-arrestin protein whose function is important for the regulation of vascular endothelial growth factor receptor 2 (VEGFR2) signaling and endothelial cell survival. Because VEGFR2 is critical for angiogenesis, we explored the role of TXNIP in VEGF-induced angiogenesis. APPROACH AND RESULTS: TXNIP knockdown inhibited VEGF-induced endothelial cell tube formation and proliferation in cultured human umbilical vein endothelial cell. To elucidate the mechanism by which TXNIP altered VEGFR2 signaling in human umbilical vein endothelial cell, we studied phosphorylation of VEGFR2, phospholipase C gamma-1 (PLCγ1), endothelial NO synthase, and Akt (known as protein kinase B). TXNIP knockdown significantly decreased phosphorylation of VEGFR2 and PLCγ1 at times >5 minutes, but phosphorylation was unchanged at 2 minutes, as was Akt and endothelial NO synthase phosphorylation. Cell-surface biotinylation assay showed that TXNIP knockdown significantly attenuated VEGFR2 internalization. These results suggested that TXNIP was required for sustained VEGFR2 signaling, which is mediated largely by internalized VEGFR2. Rab5 knockdown to inhibit the trafficking and fusion of early endosomes significantly blocked VEGF-induced VEGFR2 internalization and phosphorylation of VEGFR2 and PLCγ1. Immunofluorescence and coimmunoprecipitation showed that TXNIP was part of a complex that included Rab5 and VEGFR2. Finally, TXNIP knockdown prevented the association of VEGFR2 and Rab5. CONCLUSIONS: Our results show that TXNIP is essential for VEGFR2 internalization in Rab5 positive endosomes, which is required for endothelial cell growth and angiogenesis.

G-protein-coupled receptor kinase interacting protein-1 mediates intima formation by regulating vascular smooth muscle proliferation, apoptosis, and migration.

OBJECTIVE: The G-protein-coupled receptor kinase interacting protein-1 (GIT1) is a scaffold protein that is important for phospholipase Cγ and extracellular signal-regulated kinase 1/2 signaling induced by angiotensin II and epidermal growth factor. Because GIT1 regulates signaling by several vascular smooth muscle cell (VSMC) growth factors, we hypothesized that intima formation would be inhibited by GIT1 depletion. APPROACH AND RESULTS: Complete carotid ligation was performed on GIT1 wild-type and knockout (KO) mice. We compared changes between GIT1 wild-type and KO mice in carotid vascular remodeling, VSMC proliferation, and apoptosis in vivo and in vitro. Our data demonstrated that GIT1 deficiency significantly decreased intima formation after carotid ligation as a result of both reduced VSMC proliferation and enhanced apoptosis. To confirm the effects of GIT1 in vitro, we performed proliferation and apoptosis assays in VSMC. In mouse aortic smooth muscle cells (MASM), we found that the growth rate and [3H]-thymidine incorporation of the GIT1 KO MASM were significantly decreased compared with the wild-type MASM. Cyclin D1, which is a key cell cycle regulator, was significantly decreased in GIT1 KO cells. Serum deprivation of GIT1 KO MASM increased apoptosis 3-fold compared with wild-type MASM. Treatment of rat aortic smooth muscle cells with GIT1 small interfering RNA impaired cell migration. Both phospholipase Cγ and extracellular signal-regulated kinase 1/2 signaling were required for GIT1-dependent VSMC proliferation and migration, whereas only phospholipase Cγ was involved in GIT1-mediated VSMC apoptosis. CONCLUSIONS: GIT1 is a novel mediator of vascular remodeling by regulating VSMC proliferation, migration, and apoptosis through phospholipase Cγ and extracellular signal-regulated kinase 1/2 signaling pathways.

EVI1 acts as an inducible negative-feedback regulator of NF-κB by inhibiting p65 acetylation.

Inflammation is a hallmark of many important human diseases. Appropriate inflammation is critical for host defense; however, an overactive response is detrimental to the host. Thus, inflammation must be tightly regulated. The molecular mechanisms underlying the tight regulation of inflammation remain largely unknown. Ecotropic viral integration site 1 (EVI1), a proto-oncogene and zinc finger transcription factor, plays important roles in normal development and leukemogenesis. However, its role in regulating NF-κB-dependent inflammation remains unknown. In this article, we show that EVI1 negatively regulates nontypeable Haemophilus influenzae- and TNF-α-induced NF-κB-dependent inflammation in vitro and in vivo. EVI1 directly binds to the NF-κB p65 subunit and inhibits its acetylation at lysine 310, thereby inhibiting its DNA-binding activity. Moreover, expression of EVI1 itself is induced by nontypeable Haemophilus influenzae and TNF-α in an NF-κB-dependent manner, thereby unveiling a novel inducible negative feedback loop to tightly control NF-κB-dependent inflammation. Thus, our study provides important insights into the novel role for EVI1 in negatively regulating NF-κB-dependent inflammation, and it may also shed light on the future development of novel anti-inflammatory strategies.

Monocyte-derived Dll4 is a novel contributor to persistent systemic inflammation in HIV patients.

Background: In people living with HIV (PLWH) on combination antiretroviral therapy (cART), persistent systemic inflammation is a driving force for the progression of comorbidities, such as cardiovascular and cerebrovascular diseases. In this context, monocyte- and macrophage-related inflammation rather than T cell activation is a major cause of chronic inflammation. However, the underlying mechanism of how monocytes cause persistent systemic inflammation in PLWH is elusive. Methods and Results: In vitro, we demonstrated that lipopolysaccharides (LPS) or tumor necrosis factor alpha (TNFα), induced a robust increase of Delta-like ligand 4 (Dll4) mRNA and protein expression in human monocytes and Dll4 secretion (extracellular Dll4, exDll4) from monocytes. Enhanced membrane-bound Dll4 (mDll4) expression in monocytes triggered Notch1 activation to promote pro-inflammatory factors expression. Dll4 silencing and inhibition of Nocth1 activation diminished the LPS or TNFα -induced inflammation. exDll4 releases in response to cytokines occurred in monocytes but not endothelial cells or T cells. In clinical specimens, we found that PLWH, both male and female, on cART, showed a significant increase in mDll4 expression, activation of Dll4-Notch1 signaling, and inflammatory markers in monocytes. Although there was no sex effect on mDII4 in PLWH, plasma exDll4 was significantly elevated in males but not females compared to HIV uninfected individuals. Furthermore, exDll4 plasma levels paralleled with monocytes mDll4 in male PLWH. Circulating exDll4 was also positively associated with pro-inflammatory monocytes phenotype and negatively associated with classic monocytes phenotype in male PLWH. Conclusion: Pro-inflammatory stimuli increase Dll4 expression and Dll4-Notch1 signaling activation in monocytes and enhance monocyte proinflammatory phenotype, contributing to persistent systemic inflammation in male and female PLWH. Therefore, monocyte mDll4 could be a potential biomarker and therapeutic target of systemic inflammation. Plasma exDll4 may also play an additional role in systemic inflammation but primarily in men.

Chronic administration of hexarelin attenuates cardiac fibrosis in the spontaneously hypertensive rat.

Cardiac fibrosis is a hallmark of heart disease and plays a vital role in cardiac remodeling during heart diseases, including hypertensive heart disease. Hexarelin is one of a series of synthetic growth hormone secretagogues (GHSs) possessing a variety of cardiovascular effects via action on GHS receptors (GHS-Rs). However, the role of hexarelin in cardiac fibrosis in vivo has not yet been investigated. In the present study, spontaneously hypertensive rats (SHRs) were treated with hexarelin alone or in combination with a GHS-R antagonist for 5 wk from an age of 16 wk. Hexarelin treatment significantly reduced cardiac fibrosis in SHRs by decreasing interstitial and perivascular myocardial collagen deposition and myocardial hydroxyproline content and reducing mRNA and protein expression of collagen I and III in SHR hearts. Hexarelin treatment also increased matrix metalloproteinase (MMP)-2 and MMP-9 activities and decreased myocardial mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 in SHRs. In addition, hexarelin treatment significantly attenuated left ventricular (LV) hypertrophy, LV diastolic dysfunction, and high blood pressure in SHRs. The effect of hexarelin on cardiac fibrosis, blood pressure, and cardiac function was mediated by its receptor, GHS-R, since a selective GHS-R antagonist abolished these effects and expression of GHS-Rs was upregulated by hexarelin treatment. In summary, our data demonstrate that hexarelin reduces cardiac fibrosis in SHRs, perhaps by decreasing collagen synthesis and accelerating collagen degradation via regulation of MMPs/TIMP. Hexarelin-reduced systolic blood pressure may also contribute to this reduced cardiac fibrosis in SHRs. The present findings provided novel insights and underscore the therapeutic potential of hexarelin as an antifibrotic agent for the treatment of cardiac fibrosis.

Caspase-4/11 is critical for angiogenesis by repressing Notch1 signalling via inhibiting γ-secretase activity.

BACKGROUND AND PURPOSE: Notch1 activation mediated by γ-secretase is critical for angiogenesis. GeneCards database predicted that Caspase-4 (CASP4, with murine ortholog CASP11) interacts with presenilin-1, the catalytic core of γ-secretase. Therefore, we investigated the role of CASP4/11 in angiogenesis. EXPERIMENTAL APPROACH: In vivo, we studied the role of Casp11 in several angiogenesis mouse models using Casp11 wild-type and knockout mice. In vitro, we detected the effects of CASP4 on endothelial functions and Notch signalling by depleting or overexpressing CASP4 in human umbilical vein endothelial cells (HUVECs). The functional domain responsible for the binding of CASP4 and presenilin-1 was detected by mutagenesis and co-immunoprecipitation. KEY RESULTS: Casp11 deficiency impaired adult angiogenesis in ischaemic hindlimbs, melanoma xenografts and Matrigel plugs, but not the developmental angiogenesis of retina. Bone marrow transplantation revealed that the pro-angiogenic effect depended on CASP11 derived from non-haematopoietic cells. CASP4 expression was induced by inflammatory factors and CASP4 knockdown decreased cell viability, proliferation, migration and tube formation in HUVECs. Mechanistically, CASP4/11 deficiency increased Notch1 activation in vivo and in vitro, while CASP4 overexpression repressed Notch1 signalling in HUVECs. Moreover, CASP4 knockdown increased γ-secretase activity. The γ-Secretase inhibitor DAPT restored the effects of CASP4 siRNA on Notch1 activation and angiogenesis in HUVECs. Notably, the catalytic activity of CASP4/11 was dispensable. CASP4 directly interacted with presenilin-1 through the caspase recruitment domain (CARD). CONCLUSIONS AND IMPLICATIONS: These findings reveal a critical role of CASP4/11 in adult angiogenesis and make this molecule a promising therapeutic target for angiogenesis-related diseases in the future.

A novel peptide inhibitor of Dll4-Notch1 signalling and its pro-angiogenic functions.

BACKGROUND AND PURPOSE: The Dll4-Notch1 signalling pathway plays an important role in sprouting angiogenesis, vascular remodelling and arterial or venous specificity. Genetic or pharmacological inhibition of Dll4-Notch1 signalling leads to excessive sprouting angiogenesis. However, transcriptional inhibitors of Dll4-Notch1 signalling have not been described. EXPERIMENTAL APPROACH: We designed a new peptide targeting Notch signalling, referred to as TAT-ANK, and assessed its effects on angiogenesis. In vitro, tube formation and fibrin gel bead assay were carried out, using human umbilical vein endothelial cells (HUVECs). In vivo, Matrigel plug angiogenesis assay, a developmental retinal model and tumour models in mice were used. The mechanisms underlying TAT-ANK activity were investigated by immunochemistry, western blotting, immunoprecipitation, RT-qPCR and luciferase reporter assays. KEY RESULTS: The amino acid residues 179-191 in the G-protein-coupled receptor-kinase-interacting protein-1 (GIT1-ankyrin domain) are crucial for GIT1 binding to the Notch transcription repressor, RBP-J. We designed the peptide TAT-ANK, based on residues 179-191 in GIT1. TAT-ANK significantly inhibited Dll4 expression and Notch 1 activation in HUVECs by competing with activated Notch1 to bind to RBP-J. The analyses of biological functions showed that TAT-ANK promoted angiogenesis in vitro and in vivo by inhibiting Dll4-Notch1 signalling. CONCLUSIONS AND IMPLICATIONS: We synthesized and investigated the biological actions of TAT-ANK peptide, a new inhibitor of Notch signalling. This peptide will be of significant interest to research on Dll4-Notch1 signalling and to clinicians carrying out clinical trials using Notch signalling inhibitors. Furthermore, our findings will have important conceptual and therapeutic implications for angiogenesis-related diseases.

Reduced Notch1 Cleavage Promotes the Development of Pulmonary Hypertension.

Clinical trials of Dll4 (Delta-like 4) neutralizing antibodies (Dll4nAbs) in cancer patients are ongoing. Surprisingly, pulmonary hypertension (PH) occurs in 14% to 18% of patients treated with Dll4nAbs, but the mechanisms have not been studied. Here, PH progression was measured in mice treated with Dll4nAbs. We detected Notch signaling in lung tissues and analyzed pulmonary vascular permeability and inflammation. Notch target gene array was performed on adult human pulmonary microvascular endothelial cells (ECs) after inhibiting Notch cleavage. Similar mechanisms were studied in PH mouse models and pulmonary arterial hypertension patients. The rescue effects of constitutively activated Notch1 in vivo were also measured. We observed that Dll4nAbs induced PH in mice as indicated by significantly increased right ventricular systolic pressure, as well as pulmonary vascular and right ventricular remodeling. Mechanistically, Dll4nAbs inhibited Notch1 cleavage and subsequently impaired lung endothelial barrier function and increased immune cell infiltration in vessel walls. In vitro, Notch targeted genes' expression related to cell growth and inflammation was decreased in human pulmonary microvascular ECs after the Notch1 inactivation. In lungs of PH mouse models and pulmonary arterial hypertension patients, Notch1 cleavage was inhibited. Consistently, EC cell-cell junction was leaky, and immune cell infiltration increased in PH mouse models. Overexpression activated Notch1-attenuated progression of PH in mice. In conclusion, Dll4nAbs led to PH development in mice by impaired EC barrier function and increased immune cell infiltration through inhibition of Notch1 cleavage in lung ECs. Reduced Notch1 cleavage in lung ECs could be an underlying mechanism of PH pathogenesis.

G protein coupled receptor kinase 2 interacting protein 1 (GIT1) is a novel regulator of mitochondrial biogenesis in heart.

G-protein-coupled receptor (GPCR)-kinase interacting protein-1 (GIT1) is a multi-function scaffold protein. However, little is known about its physiological role in the heart. Here we sought to identify the cardiac function of GIT1. Global GIT1 knockout (KO) mice were generated and exhibited significant cardiac hypertrophy that progressed to heart failure. Electron microscopy revealed that the hearts of GIT1 KO mice demonstrated significant morphological abnormities in mitochondria, including decreased mitochondrial volume density, cristae density and increased vacuoles. Moreover, mitochondrial biogenesis-related gene peroxisome proliferator-activated receptor γ (PPARγ) co-activator-1α (PGC-1α), PGC-1ß, mitochondrial transcription factor A (Tfam) expression, and total mitochondrial DNA were remarkably decreased in hearts of GIT1 KO mice. These animals also had impaired mitochondrial function, as evidenced by reduced ATP production and dissipated mitochondrial membrane potential (Ψ(m)) in adult cardiomyocytes. Concordant with these mitochondrial observations, GIT1 KO mice showed enhanced cardiomyocyte apoptosis and cardiac dysfunction. In conclusion, our findings identify GIT1 as a new regulator of mitochondrial biogenesis and function, which is necessary for postnatal cardiac maturation.

Phosphorylation of G protein-coupled receptor kinase 2-interacting protein 1 tyrosine 392 is required for phospholipase C-gamma activation and podosome formation in vascular smooth muscle cells.

OBJECTIVE: Podosomes, which are actin-rich structures, contribute to cell motility, matrix remodeling, and tissue remodeling. We have shown that G protein-coupled receptor kinase 2-interacting protein 1 (GIT1) colocalizes with podosomes and is important in podosome formation in endothelial cells. Src stimulates GIT1 tyrosine phosphorylation, which is critical for phospholipase C-γ (PLCγ) activation. In this study, we identified specific GIT1 tyrosines required for PLCγ activation and podosome formation in vascular smooth muscle cells (VSMC). METHODS AND RESULTS: We used phorbol 12,13-dibutyrate (PDBU) to induce podosomes in A7r5 VSMC. GIT1 colocalized with podosomes and GIT1 knockdown using short interfering RNA significantly reduced podosome formation. PDBU stimulated GIT1 tyrosine phosphorylation. GIT1 tyrosine phosphorylation was dramatically decreased in SYF-/- cells, and it was also reduced by pretreatment with the protein kinase C (PKC) and Src inhibitors, suggesting that GIT1 phosphorylation was dependent on PKC and Src. By mutation analysis of multiple tyrosines, we found that PDBU specifically increased GIT1-Y392 phosphorylation. Overexpression of GIT1 (Y392F) but not of GIT1 (Y321F) decreased PDBU-mediated PLCγ activation and podosome formation without effect on extracellular signal-regulated kinase 1/2 activation. Additionally, we provide evidence that GIT1 knockout VSMC have markedly fewer podosomes on PDBU treatment compared with wild-type VSMC. These data show that GIT1 is a key regulator of podosome formation in VSMC. CONCLUSIONS: In conclusion, our data suggest that GIT1-Y392 phosphorylation is critical for PDBU-induced podosome formation by regulating PLCγ activation. We propose that specific signaling modules are assembled in a GIT1 phosphotyrosine-dependent manner as exemplified by PLCγ activation versus extracellular signal-regulated kinase 1/2 activation.

G-protein-coupled receptor kinase interacting protein-1 is required for pulmonary vascular development.

BACKGROUND: The G-protein-coupled receptor kinase interacting protein-1 (GIT1) is a multidomain scaffold protein that participates in many cellular functions including receptor internalization, focal adhesion remodeling, and signaling by both G-protein-coupled receptors and tyrosine kinase receptors. However, there have been no in vivo studies of GIT1 function to date. METHODS AND RESULTS: To determine essential functions of GIT1 in vivo, we generated a traditional GIT1 knockout mouse. GIT1 knockout mice exhibited approximately 60% perinatal mortality. Pathological examination showed that the major abnormality in GIT1 knockout mice was impaired lung development characterized by markedly reduced numbers of pulmonary blood vessels and increased alveolar spaces. Given that vascular endothelial growth factor (VEGF) is essential for pulmonary vascular development, we investigated the role of GIT1 in VEGF signaling in the lung and cultured endothelial cells. Because activation of phospholipase-Cgamma (PLCgamma) and extracellular signal-regulated kinases 1/2 (ERK1/2) by angiotensin II requires GIT1, we hypothesized that GIT1 mediates VEGF-dependent pulmonary angiogenesis by modulating PLCgamma and ERK1/2 activity in endothelial cells. In cultured endothelial cells, knockdown of GIT1 decreased VEGF-mediated phosphorylation of PLCgamma and ERK1/2. PLCgamma and ERK1/2 activity in lungs from GIT1 knockout mice was reduced postnatally. CONCLUSIONS: Our data support a critical role for GIT1 in pulmonary vascular development by regulating VEGF-induced PLCgamma and ERK1/2 activation.

GIT1 mediates VEGF-induced podosome formation in endothelial cells: critical role for PLCgamma.

OBJECTIVE: We and others showed that tyrosine kinase receptors (TKRs) such as the epidermal growth factor receptor stimulate G protein-coupled receptor (GPCR) kinase-interacting protein 1 (GIT1) phosphorylation via c-Src, which is required for phospholipase C-gamma (PLCgamma) activation, indicating that GIT1 participates in TKR signaling. VEGF is the most important TKR in endothelial cells (ECs); essential for cell survival, migration, and angiogenesis. Podosomes, actin-rich structures, were found to contribute to EC migration, tissue invasion, and matrix remodeling, suggesting a role for podosomes in angiogenesis. Because GIT1 is a substrate of c-Src, and podosome formation is c-Src dependent, we hypothesized that GIT1 plays an important role in VEGF-induced EC podosome formation and cell migration. METHODS AND RESULTS: Exposure of ECs to VEGF for 30 minutes stimulated GIT1 colocalization with podosomes. Depletion of GIT1 by siRNA significantly decreased VEGF-induced podosome formation. A key role for PLCgamma was suggested by several experiments. Double staining PLCgamma and actin showed colocalization of PLCgamma with podosomes. Podosome formation was dramatically reduced by PLCgamma inhibitor U73122, Src inhibitor PP2, or expression of dominant negative small GTPases. Therefore, VEGF-induced EC podosome formation is dependent on Src, GIT1, PLCgamma, and small GTPases. In addition, matrix metalloprotease 2 (MMP2) and MT-MMP1 were detected at sites of VEGF-induced podosomes. Depletion of GIT1 by siRNA also significantly inhibited VEGF-induced MMP2 activation and extracellular matrix (ECM) degradation. Therefore, GIT1 mediates VEGF-induced matrix metalloproteinase (MMP) activation and ECM degradation by regulating podosome formation. Finally, depletion of GIT1 by siRNA significantly decreased VEGF-induced cell migration. CONCLUSIONS: These data indicate that GIT1 is an essential mediator for VEGF-induced EC podosome formation and cell migration via PLCgamma.

Endothelial-to-Mesenchymal Transition and Inflammation Play Key Roles in Cyclophilin A-Induced Pulmonary Arterial Hypertension.

Oxidative stress and inflammation play key roles in development of pulmonary arterial hypertension (PAH). We previously reported that an endothelial cell (EC)-specific cyclophilin A overexpression mouse developed many characteristics of PAH. In other models of cardiovascular disease, cyclophilin A stimulates smooth muscle proliferation and vascular inflammation, but mechanisms responsible for PAH have not been defined. In particular, the contribution of endothelial-to-mesenchymal transition in cyclophilin A-mediated PAH has not been studied. We identified increased levels of cyclophilin A in endothelial and neointimal cells of pulmonary arteries in patients with PAH and animal pulmonary hypertension models. In the EC-specific cyclophilin A overexpression mouse that exhibited features characteristic of PAH, lineage tracing showed high level expression of mesenchymal markers in pulmonary ECs. A significant number of mesenchymal cells in media and perivascular regions of pulmonary arterioles and alveoli were derived from ECs. Pulmonary ECs isolated from these mice showed phenotypic changes characteristic of endothelial-to-mesenchymal transition in culture. Cultured pulmonary ECs stimulated with extracellular cyclophilin A and acetylated cyclophilin A demonstrated functional changes associated with endothelial-to-mesenchymal transition such as increased cytokine release, migration, proliferation, and mitochondrial dysfunction. Acetylated cyclophilin A stimulated greater increases for most features of endothelial-to-mesenchymal transition. In conclusion, extracellular cyclophilin A (especially acetylated form) contributes to PAH by mechanisms involving increased endothelial-to-mesenchymal transition, cytokine release, EC migration, proliferation, and mitochondrial dysfunction; strengthening the basis for studying cyclophilin A inhibition as a therapy for PAH.