Endothelial phosphoinositide 3-kinase-ß inactivation confers protection from immune-mediated vascular injury.

Heart transplant and recipient survival are limited by immune cell-mediated injury of the graft vasculature. We examined the role of the phosphoinositide 3-kinase-ß (PI3Kß) isoform in endothelial cells (EC) during coronary vascular immune injury and repair in mice. In minor histocompatibility-antigen mismatched allogeneic heart grafts, a robust immune response was mounted to each wild-type, PI3Kß inhibitor-treated, or endothelial-selective PI3Kß knockout (ECßKO) graft transplanted to wild-type recipients. However, microvascular EC loss and progressive occlusive vasculopathy only developed in control, but not PI3Kß-inactivated hearts. We observed a delay in inflammatory cell infiltration of the ECßKO grafts, particularly in the coronary arteries. Surprisingly, this was accompanied by an impaired display of proinflammatory chemokine and adhesion molecules by the ECßKO ECs. In vitro, tumor necrosis factor α-stimulated endothelial ICAM1 and VCAM1 expression was blocked by PI3Kß inhibition or RNA interference. Selective PI3Kß inhibition also blocked tumor necrosis factor α-stimulated degradation of inhibitor of nuclear factor kappa Bα and nuclear translocation of nuclear factor kappa B p65 in EC. These data identify PI3Kß as a therapeutic target to reduce vascular inflammation and injury.

Loss of apelin blocks the emergence of sprouting angiogenesis in experimental tumors.

Angiogenesis inhibitor drugs targeting vascular endothelial growth factor (VEGF) signaling to the endothelial cell (EC) are used to treat various cancer types. However, primary or secondary resistance to therapy is common. Clinical and pre-clinical studies suggest that alternative pro-angiogenic factors are upregulated after VEGF pathway inhibition. Therefore, identification of alternative pro-angiogenic pathway(s) is critical for the development of more effective anti-angiogenic therapy. Here we study the role of apelin as a pro-angiogenic G-protein-coupled receptor ligand in tumor growth and angiogenesis. We found that loss of apelin in mice delayed the primary tumor growth of Lewis lung carcinoma 1 and B16F10 melanoma when combined with the VEGF receptor tyrosine kinase inhibitor, sunitinib. Targeting apelin in combination with sunitinib markedly reduced the tumor vessel density, and decreased microvessel remodeling. Apelin loss reduced angiogenic sprouting and tip cell marker gene expression in comparison to the sunitinib-alone-treated mice. Single-cell RNA sequencing of tumor EC demonstrated that the loss of apelin prevented EC tip cell differentiation. Thus, apelin is a potent pro-angiogenic cue that supports initiation of tumor neovascularization. Together, our data suggest that targeting apelin may be useful as adjuvant therapy in combination with VEGF signaling inhibition to inhibit the growth of advanced tumors.

FGD5 regulates endothelial cell PI3 kinase-ß to promote neo-angiogenesis.

Angiogenesis is required in embryonic development and tissue repair in the adult. Vascular endothelial growth factor (VEGF) initiates angiogenesis, and VEGF or its receptor is targeted therapeutically to block pathological angiogenesis. Additional pro-angiogenic cues, such as CXCL12 acting via the CXCR4 receptor, co-operate with VEGF/VEGFR2 to cue vascular patterning. We studied the role of FGD5, an endothelial Rho GTP/GDP exchange factor (RhoGEF), to regulate CXCR4-dependent signals in the endothelial cell (EC). Patient-derived renal cell carcinomas produce a complex milieu of growth factors that stimulated sprouting angiogenesis and endothelial tip cell differentiation ex vivo that was blocked by EC FGD5 loss. In a simplified model, CXCL12 augmented sprouting and tip gene expression under conditions where VEGF was limiting. CXCL12-stimulated tip cell differentiation was dependent on PI3 kinase (PI3K)-ß activity. Knockdown of EC FGD5 abolished CXCR4 signaling to PI3K-ß and Akt. Further, inhibition of Rac1, a Rho GTPase required for PI3K-ß activity, recapitulated the signaling defects of FGD5 deficiency, suggesting that FGD5 may regulate PI3K-ß activity through Rac1. Overexpression of a RhoGEF deficient, Dbl domain-deleted FGD5 mutant reduced CXCL12-stimulated Akt phosphorylation and failed to rescue PI3K signaling in native FGD5-deficient EC, indicating that FGD5 RhoGEF activity is required for FDG5 function. Endothelial expression of mutant PI3K-ß with an inactivated Rho binding domain confirmed that CXCL12-stimulated PI3K activity in EC requires Rac1-GTP co-regulation. Together, this data identify the role of FGD5 to generate Rac1-GTP to regulate pro-angiogenic CXCR4-dependent PI3K-ß signaling in EC. Inhibition of FGD5 activity may complement current angiogenesis inhibitor drugs.

Gene Expression Profiling in Kidney Transplants with Immune Checkpoint Inhibitor-Associated Adverse Events.

BACKGROUND AND OBJECTIVES: Immune checkpoint inhibitors are increasingly used to treat various malignancies, but their application in patients with kidney transplants is complicated by high allograft rejection rates. Immune checkpoint inhibitor-associated rejection is a novel, poorly understood entity demonstrating overlapping histopathologic features with immune checkpoint inhibitor-associated acute interstitial nephritis, which poses a challenge for diagnosis and clinical management. We sought to improve the understanding of these entities through biopsy-based gene expression analysis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: NanoString was used to measure and compare the expression of 725 immune-related genes in 75 archival kidney biopsies, including a 25-sample discovery cohort comprising pure T cell-mediated rejection and immune checkpoint inhibitor-associated acute interstitial nephritis and an independent 50-sample validation cohort comprising immune checkpoint inhibitor-associated acute interstitial nephritis, immune checkpoint inhibitor-associated T cell-mediated rejection, immune checkpoint inhibitor-associated crescentic GN, drug-induced acute interstitial nephritis, BK virus nephropathy, and normal biopsies. RESULTS: Significant molecular overlap was observed between immune checkpoint inhibitor-associated acute interstitial nephritis and T cell-mediated rejection. Nevertheless, IFI27, an IFN-α-induced transcript, was identified and validated as a novel biomarker for differentiating immune checkpoint inhibitor-associated T cell-mediated rejection from immune checkpoint inhibitor-associated acute interstitial nephritis (validation cohort: P<0.001, area under the receiver operating characteristic curve =100%, accuracy =86%). Principal component analysis revealed heterogeneity in inflammatory gene expression patterns within sample groups; however, immune checkpoint inhibitor-associated T cell-mediated rejection and immune checkpoint inhibitor-associated acute interstitial nephritis both demonstrated relatively more molecular overlap with drug-induced acute interstitial nephritis than T cell-mediated rejection, suggesting potential dominance of hypersensitivity mechanisms in these entities. CONCLUSIONS: These results indicate that, although there is significant molecular similarity between immune checkpoint inhibitor-associated rejection and acute interstitial nephritis, biopsy-based measurement of IFI27 gene expression represents a potential biomarker for differentiating these entities.

Pharmacological and cell-specific genetic PI3Kα inhibition worsens cardiac remodeling after myocardial infarction.

BACKGROUND: PI3Kα (Phosphoinositide 3-kinase α) regulates multiple downstream signaling pathways controlling cell survival, growth, and proliferation and is an attractive therapeutic target in cancer and obesity. The clinically-approved PI3Kα inhibitor, BYL719, is in further clinical trials for cancer and overgrowth syndrome. However, the potential impact of PI3Kα inhibition on the heart and following myocardial infarction (MI) is unclear. We aim to determine whether PI3Kα inhibition affects cardiac physiology and post-MI remodeling and to elucidate the underlying molecular mechanisms. METHODS AND RESULTS: Wildtype (WT) 12-wk old male mice receiving BYL719 (daily, p.o.) for 10 days showed reduction in left ventricular longitudinal strain with normal ejection fraction, weight loss, mild cardiac atrophy, body composition alteration, and prolonged QTC interval. RNASeq analysis showed gene expression changes in multiple pathways including extracellular matrix remodeling and signaling complexes. After MI, both p110α and phospho-Akt protein levels were increased in human and mouse hearts. Pharmacological PI3Kα inhibition aggravated cardiac dysfunction and resulted in adverse post-MI remodeling, with increased apoptosis, elevated inflammation, suppressed hypertrophy, decreased coronary blood vessel density, and inhibited Akt/GSK3ß/eNOS signaling. Selective genetic ablation of PI3Kα in endothelial cells was associated with worsened post-MI cardiac function and reduced coronary blood vessel density. In vitro, BYL719 suppressed Akt/eNOS activation, cell viability, proliferation, and angiogenic sprouting in coronary and human umbilical vein endothelial cells. Cardiomyocyte-specific genetic PI3Kα ablation resulted in mild cardiac systolic dysfunction at baseline. After MI, cardiac function markedly deteriorated with increased mortality concordant with greater apoptosis and reduced hypertrophy. In isolated adult mouse cardiomyocytes, BYL719 decreased hypoxia-associated activation of Akt/GSK3ß signaling and cell survival. CONCLUSIONS: PI3Kα is required for cell survival (endothelial cells and cardiomyocytes) hypertrophic response, and angiogenesis to maintain cardiac function after MI. Therefore, PI3Kα inhibition that is used as anti-cancer treatment, can be cardiotoxic, especially after MI.

Fgd5 is a Rac1-specific Rho GEF that is selectively inhibited by aurintricarboxylic acid.

Rho proteins are signalling molecules that control cellular dynamics, movement and morphological changes. They are activated by Rho guanine-nucleotide exchange factors (Rho GEFs) that transduce upstream signals into Rho-mediated activation of downstream processes. Fgd5 is a Rho GEF involved in angiogenesis and its target Rho protein for this process has been linked to Cdc42 activation. Here, we examined the function of purified Fgd5, specifically, which Rho proteins it activates and pinpoint the structural domains required for enzymatic activity. Using a GEF enzyme assay, we found that purified Fgd5 showed preferential activation of Rac1 and direct binding of Rac1 in pull-down and co-immunoprecipitation assays. Structural comparisons showed that the Fgd5 DH domain is highly similar to the Rac1 GEF, TrioN, supporting a role for Fgd5 as a Rac1 GEF. Compounds that bind to purified Fgd5 DH-PH protein were identified by screening a small molecule library via surface plasmon resonance. The effects of eleven ligands were further examined for their ability to inhibit the Fgd5 GEF enzymatic activity and Rac1 interaction. From these studies, we found that the compound aurintricarboxylic acid, and to a lesser extent mitoxantrone dihydrochloride, inhibited both Fgd5 GEF activation of Rac1 and their interaction. Aurintricarboxylic acid had no effect on the activity or binding of the Rac1 GEF, TrioN, thus demonstrating the feasibility of selectively disrupting Rho GEF activators. Abbreviations: a.a.: amino acid; ATA: aurintricarboxylic acid; DH: Dbl homology; DOCK: dictator of cytokinesis; Fgd: faciogenital dysplasia; GEF: guanine-nucleotide exchange factor; GST: glutathione S-transferase; LOPAC: library of pharmacologically active compounds; PH: pleckstrin homology; PDB: protein data bank; s.e.m.: standard error of the mean; SPR: surface plasmon resonance.

Inactivation of endothelial cell phosphoinositide 3-kinase ß inhibits tumor angiogenesis and tumor growth.

Angiogenesis inhibitors, such as the receptor tyrosine kinase (RTK) inhibitor sunitinib, target vascular endothelial growth factor (VEGF) signaling in cancers. However, only a fraction of patients respond, and most ultimately develop resistance to current angiogenesis inhibitor therapies. Activity of alternative pro-angiogenic growth factors, acting via RTK or G-protein coupled receptors (GPCR), may mediate VEGF inhibitor resistance. The phosphoinositide 3-kinase (PI3K)ß isoform is uniquely coupled to both RTK and GPCRs. We investigated the role of endothelial cell (EC) PI3Kß in tumor angiogenesis. Pro-angiogenic GPCR ligands were expressed by patient-derived renal cell carcinomas (PD-RCC), and selective inactivation of PI3Kß reduced PD-RCC-stimulated EC spheroid sprouting. EC-specific PI3Kß knockout (ΕC-ßKO) in mice potentiated the sunitinib-induced reduction in subcutaneous growth of LLC1 and B16F10, and lung metastasis of B16F10 tumors. Compared to single-agent sunitinib treatment, tumors in sunitinib-treated ΕC-ßKO mice showed a marked decrease in microvessel density, and reduced new vessel formation. The fraction of perfused mature tumor microvessels was increased in ΕC-ßKO mice suggesting immature microvessels were most sensitive to combined sunitinib and PI3Kß inactivation. Taken together, EC PI3Kß inactivation with sunitinib inhibition reduces microvessel turnover and decreases heterogeneity of the tumor microenvironment, hence PI3Kß inhibition may be a useful adjuvant antiangiogenesis therapy with sunitinib.

Apelin directs endothelial cell differentiation and vascular repair following immune-mediated injury.

Sustained, indolent immune injury of the vasculature of a heart transplant limits long-term graft and recipient survival. This injury is mitigated by a poorly characterized, maladaptive repair response. Vascular endothelial cells respond to proangiogenic cues in the embryo by differentiation to specialized phenotypes, associated with expression of apelin. In the adult, the role of developmental proangiogenic cues in repair of the established vasculature is largely unknown. We found that human and minor histocompatibility-mismatched donor mouse heart allografts with alloimmune-mediated vasculopathy upregulated expression of apelin in arteries and myocardial microvessels. In vivo, loss of donor heart expression of apelin facilitated graft immune cell infiltration, blunted vascular repair, and worsened occlusive vasculopathy in mice. In vitro, an apelin receptor agonist analog elicited endothelial nitric oxide synthase activation to promote endothelial monolayer wound repair and reduce immune cell adhesion. Thus, apelin acted as an autocrine growth cue to sustain vascular repair and mitigate the effects of immune injury. Treatment with an apelin receptor agonist after vasculopathy was established markedly reduced progression of arterial occlusion in mice. Together, these initial data identify proangiogenic apelin as a key mediator of coronary vascular repair and a pharmacotherapeutic target for immune-mediated injury of the coronary vasculature.

Regulation of von Willebrand Factor Gene in Endothelial Cells That Are Programmed to Pluripotency and Differentiated Back to Endothelial Cells.

Endothelial cells play a central role in physiological function and pathophysiology of blood vessels in health and disease. However, the molecular mechanism that establishes the endothelial phenotype, and contributes to its signature cell type-specific gene expression, is not yet understood. We studied the regulation of a highly endothelial-specific gene, von Willebrand factor (VWF), in induced pluripotent stem cells generated from primary endothelial cells (human umbilical vein endothelial cells [HUVEC] into a pluripotent state [HiPS]) and subsequently differentiated back into endothelial cells. This allowed us to explore how VWF expression is regulated when the endothelial phenotype is revoked (endothelial cells to HiPS), and re-established (HiPS back to endothelial cells [EC-Diff]). HiPS were generated from HUVECs, their pluripotency established, and then differentiated back to endothelial cells. We established phenotypic characteristics and robust angiogenic function of EC-Diff. Gene array analyses, VWF chromatin modifications, and transacting factors binding assays were performed on the three cell types (HUVEC, HiPS, and EC-Diff). The results demonstrated that generally cohorts of transacting factors that function as transcriptional activators, and those that contribute to histone acetylation and DNA demethylation, were significantly decreased in HiPS compared with HUVECs and EC-Diff. In contrast, there were significant increases in the gene expression levels of epigenetic modifiers that function as methyl transferases in HiPS compared with endothelial cells. The results demonstrated that alterations in chromatin modifications of the VWF gene, in addition to expression and binding of transacting factors that specifically function as activators, are responsible for establishing endothelial specific regulation of the VWF gene. Stem Cells 2019;37:542-554.

Structure and function of the Fgd family of divergent FYVE domain proteins 1.

FYVE domains are highly conserved protein modules that typically bind phosphatidylinositol 3-phosphate (PI3P) on the surface of early endosomes. Along with pleckstrin homology (PH) and phox homology (PX) domains, FYVE domains are the principal readers of the phosphoinositide (PI) code that mediate specific recognition of eukaryotic organelles. Of all the human FYVE domain containing proteins, those within the faciogenital dysplasia (Fgd) subfamily are particularly divergent and couple with GTPases to exert unique cellular functions. The subcellular distributions and functions of these evolutionarily conserved signal transducers, which also include Dbl homology (DH) and two PH domains, are discussed here to better understand the biological range of processes that such multidomain proteins engage in. Determinants of their various functions include specific multidomain architectures, posttranslational modifications including PIP stops that have been discovered in sorting nexins, PI recognition motifs, and phospholipid-binding surfaces as defined by the Membrane Optimal Docking Area (MODA) program. How these orchestrate Fgd function remains unclear but has implications for developmental diseases including Aarskog-Scott syndrome, which is also known as faciogenital dysplasia, and forms of cancer that are associated with mutations and amplifications of Fgd genes.

Endothelial and cardiomyocyte PI3Kß divergently regulate cardiac remodelling in response to ischaemic injury.

AIMS: Cardiac remodelling in the ischaemic heart determines prognosis in patients with ischaemic heart disease (IHD), while enhancement of angiogenesis and cell survival has shown great potential for IHD despite translational challenges. Phosphoinositide 3-kinase (PI3K)/Akt signalling pathways play a critical role in promoting angiogenesis and cell survival. However, the effect of PI3Kß in the ischaemic heart is poorly understood. This study investigates the role of endothelial and cardiomyocyte (CM) PI3Kß in post-infarct cardiac remodelling. METHODS AND RESULTS: PI3Kß catalytic subunit-p110ß level was increased in infarcted murine and human hearts. Using cell type-specific loss-of-function approaches, we reported novel and distinct actions of p110ß in endothelial cells (ECs) vs. CMs in response to myocardial ischaemic injury. Inactivation of endothelial p110ß resulted in marked resistance to infarction and adverse cardiac remodelling with decreased mortality, improved systolic function, preserved microvasculature, and enhanced Akt activation. Cultured ECs with p110ß knockout or inhibition displayed preferential PI3Kα/Akt/endothelial nitric oxide synthase signalling that consequently promoted protective signalling and angiogenesis. In contrast, mice with CM p110ß-deficiency exhibited adverse post-infarct ventricular remodelling with larger infarct size and deteriorated cardiac function, which was due to enhanced susceptibility of CMs to ischaemia-mediated cell death. Disruption of CM p110ß signalling compromised nuclear p110ß and phospho-Akt levels leading to perturbed gene expression and elevated pro-cell death protein levels, increasing the susceptibility to CM death. A similar divergent response of PI3Kß endothelial and CM mutant mice was seen using a model of myocardial ischaemia-reperfusion injury. CONCLUSION: These data demonstrate novel, differential, and cell-specific functions of PI3Kß in the ischaemic heart. While the loss of endothelial PI3Kß activity produces cardioprotective effects, CM PI3Kß is protective against myocardial ischaemic injury.

FGD5 Regulates VEGF Receptor-2 Coupling to PI3 Kinase and Receptor Recycling.

OBJECTIVE: VEGF (vascular endothelial growth factor-A) signaling to the endothelial cell (EC) through VEGFR2 (VEGF receptor-2) is the principal cue driving new blood vessel formation. FGD5 (faciogenital dysplasia-5)-a Rho-family guanine nucleotide exchange factor-is selectively expressed in EC. Deficiency of FGD5 is embryonically lethal in mice and perturbs angiogenesis and VEGF signal transduction. However, the mechanism of FGD5 regulation of VEGF signaling is poorly understood. APPROACH AND RESULTS: Angiogenic sprouting and EC cytoskeletal remodeling were evaluated in a 3-dimensional in vitro model. We examined the subcellular localization of FGD5 and VEGFR2 in EC by immunofluorescent staining and studied the association by immunoprecipitation. FGD5 deficiency reduced the number of angiogenic sprouts and tip cell filopodia by ≈80% and ≈70%, respectively. These defects were accompanied by downregulation of the expression of tip cell-specific markers. FGD5 inactivation led to a decrease in EC migration and early protrusion (lamellipodia) formation. In resting and VEGF-stimulated EC, FGD5 forms a complex with VEGFR2 and was enriched at the leading edge of the cell and among endosomes. FGD5 loss reduced mTORC2 (mammalian target of rapamycin complex-2)/Akt-dependent cortactin activation downstream of VEGFR2 but did not alter VEGFR2 plasma membrane expression, Y1175 phosphorylation, or endocytosis. However, FGD5 loss decreased endosomal VEGFR2 coupling to phosphoinositide-3 kinase and diverted VEGFR2 to lysosomal degradation. CONCLUSIONS: FGD5 regulates VEGFR2 retention in recycling endosomes and coupling to PI3 (phosphoinositide-3) kinase/mTORC2-dependent cytoskeletal remodeling.

Myocardial overexpression of TIMP3 after myocardial infarction exerts beneficial effects by promoting angiogenesis and suppressing early proteolysis.

Myocardial infarction (MI) results in loss of cardiomyocytes, adverse extracellular matrix (ECM) and structural remodeling, and left ventricular (LV) dilation and dysfunction. Tissue inhibitors of metalloproteinase (TIMPs) inhibit matrix metalloproteinases (MMPs), the main regulators of ECM turnover. TIMPs also have MMP-independent functions. TIMP3 levels are reduced in the heart within 24 h of MI in mice. We investigated if overexpression of TIMP3 post-MI limits adverse remodeling and LV dilation and dysfunction. MI was induced by left anterior descending coronary artery ligation in 10- to 12-wk-old male C57BL/6J mice, and adenoviral constructs expressing human (h)TIMP3 (Ad-hTIMP3) or no TIMP (Ad-Null) were injected in the peri-infarct zone (5.4 × 107 plaque-forming units/heart, 5 injections/heart). Cardiac function assessed by echocardiography showed improved LV physiology and reduced LV dilation after TIMP3 overexpression compared with the Ad-Null-MI group. Post-MI adverse remodeling was attenuated in the Ad-hTIMP3-MI group, as assessed by greater cardiomyocyte density, less infarct expansion, and ECM disruption. TIMP3 overexpression blunted the early rise in proteolytic activities post-MI. A higher density of coronary arteries and a greater number of proliferating endothelial cells were detected in the infarct and peri-infarct regions in the Ad-hTIMP3-MI group compared with the Ad-Null-MI group. In vitro three-dimensional angiogenesis assay confirmed that recombinant TIMP3 promotes angiogenesis in human endothelial cells, although biphasically and in a dose-dependent manner. Intriguingly, overexpression of Ad-hTIMP3 at 10-fold higher concentration had no beneficial effects, consistent with antiangiogenic effects of TIMP3 at higher doses. In conclusion, optimal overexpression of TIMP3 can be a promising therapeutic approach to limit adverse post-MI remodeling by dually inhibiting early proteolysis and promoting angiogenesis.NEW & NOTEWORTHY Here, we report that tissue inhibitor of metalloproteinase 3 overexpression after myocardial infarction improves myocardial structural remodeling and function by promoting angiogenesis and inhibiting early proteolysis. This demonstrates the therapeutic potential of preserving the local balance of tissue inhibitor of metalloproteinase 3 in the heart given its diverse functions in modulating different processes involved in the adverse postmyocardial infarction remodeling.

Angiotensin-Converting Enzyme 2 Metabolizes and Partially Inactivates Pyr-Apelin-13 and Apelin-17: Physiological Effects in the Cardiovascular System.

Apelin peptides mediate beneficial effects on the cardiovascular system and are being targeted as potential new drugs. However, apelin peptides have extremely short biological half-lives, and improved understanding of apelin peptide metabolism may lead to the discovery of biologically stable analogues with therapeutic potential. We examined the ability of angiotensin-converting enzyme 2 (ACE2) to cleave and inactivate pyr-apelin 13 and apelin 17, the dominant apelin peptides. Computer-assisted modeling shows a conserved binding of pyr-apelin 13 and apelin 17 to the ACE2 catalytic site. In ACE2 knockout mice, hypotensive action of pyr-apelin 13 and apelin 17 was potentiated, with a corresponding greater elevation in plasma apelin levels. Similarly, pharmacological inhibition of ACE2 potentiated the vasodepressor action of apelin peptides. Biochemical analysis confirmed that recombinant human ACE2 can cleave pyr-apelin 13 and apelin 17 efficiently, and apelin peptides are degraded slower in ACE2-deficient plasma. The biological relevance of ACE2-mediated proteolytic processing of apelin peptides was further supported by the reduced potency of pyr-apelin 12 and apelin 16 on the activation of signaling pathways and nitric oxide production from endothelial cells. Importantly, although pyr-apelin 13 and apelin 17 rescued contractile function in a myocardial ischemia-reperfusion model, ACE2 cleavage products, pyr-apelin 12 and 16, were devoid of these cardioprotective effects. We designed and synthesized active apelin analogues that were resistant to ACE2-mediated degradation, thereby confirming that stable apelin analogues can be designed as potential drugs. We conclude that ACE2 represents a major negative regulator of apelin action in the vasculature and heart.

Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis.

Tumor neovascularization is targeted by inhibition of vascular endothelial growth factor (VEGF) or the receptor to prevent tumor growth, but drug resistance to angiogenesis inhibition limits clinical efficacy. Inhibition of the phosphoinositide 3 kinase pathway intermediate, mammalian target of rapamycin (mTOR), also inhibits tumor growth and may prevent escape from VEGF receptor inhibitors. mTOR is assembled into two separate multi-molecular complexes, mTORC1 and mTORC2. The direct effect of mTORC2 inhibition on the endothelium and tumor angiogenesis is poorly defined. We used pharmacological inhibitors and RNA interference to determine the function of mTORC2 versus Akt1 and mTORC1 in human endothelial cells (EC). Angiogenic sprouting, EC migration, cytoskeleton re-organization, and signaling events regulating matrix adhesion were studied. Sustained inactivation of mTORC1 activity up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and VEGF. mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt1. Endothelial mTORC2 regulates angiogenesis, in part by regulation of EC focal adhesion kinase activity, matrix adhesion, and cytoskeletal remodeling, independent of Akt/mTORC1.

Reprogramming of HUVECs into induced pluripotent stem cells (HiPSCs), generation and characterization of HiPSC-derived neurons and astrocytes.

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for "patient-matched" cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their treatments.

PI3Kα is essential for the recovery from Cre/tamoxifen cardiotoxicity and in myocardial insulin signalling but is not required for normal myocardial contractility in the adult heart.

AIMS: Genetic mouse models have yielded conflicting conclusions about the role of PI3Kα in heart physiology: specifically, the question of whether PI3Kα has a direct role in regulating myocardial contractility. This has led to concerns that PI3K inhibitors currently in clinical trials for cancer may potentiate cardiotoxicity. Here we seek to clarify the role of PI3Kα in normal heart physiology and investigate changes in related signalling pathways. METHODS AND RESULTS: Targeted deletion of PI3Kα and PI3Kß in the heart with a tamoxifen-dependent Cre recombinase transgene caused transient heart dysfunction in all genotypes, but only PI3Kα deletion prevented functional recovery. Reduction in tamoxifen dosing allowed for maintained gene deletion without any cardiomyopathy, possibly through activation of survival signalling through the related ERK pathway. Similarly, mice with PI3Kα deletion induced by constitutively active Cre recombinase had normal heart function. Insulin-mediated activation of Akt, a marker of PI3Kα activity, was impaired with increased ERK1/2 activation in PI3Kα mutant hearts. Pharmacological inhibition of PI3Kα with BYL-719 also caused impaired insulin signalling in murine and human cardiomyocytes as well as in vivo in mice, with increased fasting blood glucose levels, but did not affect myocardial contractility as determined by echocardiography and invasive pressure-volume loop analysis. CONCLUSION: Our results show that PI3Kα does not directly regulate myocardial contractility, but is required for recovery from tamoxifen/Cre toxicity. The important role for PI3Kα in insulin signalling and recovery from tamoxifen/Cre toxicity justifies caution when using PI3Kα inhibitors in combination with other cardiovascular comorbidities and cardiotoxic compounds in cancer patients.

Phosphoinositide 3-kinase ß mediates microvascular endothelial repair of thrombotic microangiopathy.

Thrombotic microangiopathy (TMA) commonly involves injury of kidney glomerular endothelial cells (ECs) and fibrin occlusion of the capillaries. The mechanisms underlying repair of the microvasculature and recovery of kidney function are poorly defined. In the developing vasculature, the phosphoinositide 3-kinase (PI3K) α isoform integrates many growth factor cues. However, the role of individual isoforms in repair of the established vasculature is unclear. We found that postnatal endothelial deletion of PI3Kß sensitizes mice to lethal acute kidney failure after TMA injury. In vitro, PI3Kß-deficient ECs show reduced angiogenic invasion of fibrin matrix with unaltered sensitivity to proapoptotic stress compared with wild-type ECs. This correlates with decreased expression of the EC tip cell markers apelin and Dll4 and is associated with a reduction in migration and proliferation. In vivo, PI3Kß-knockdown ECs are deficient in assembly of microvessel-like structures. These data identify a critical role for endothelial PI3Kß in microvascular repair following injury.

Experimental glomerular endothelial injury in vivo.

The microvascular endothelium of the kidney glomerulus is injured in Shiga-like toxigenic bacterial infection, genetic or acquired loss of complement regulatory protein function, and allo-immune responses of solid-organ or bone marrow transplantation. Existing models of diseases with glomerular endothelial cell (EC) injury, collectively grouped as thrombotic microangiopathies, are problematic, impeding investigation of the mechanisms of microvascular defense and repair. To develop a model of glomerular endothelial injury in the mouse, we conjugated the M. oreades lectin to the cytotoxin, saporin, (LS) to selectively injure the glomerular endothelium. Injury of the microvasculature was evaluated by light, immunofluorescence, and electron microscopy, and by quantitative RT-PCR of cell-type specific transcripts. Renal function was evaluated by quantitation of serum creatinine. The toxin conjugate induced apoptosis of microvascular ECs in vitro, and subtle histologic features of thrombotic microangiopathy in vivo that were enhanced by co-injection of 50 µg/kg LPS. Among LS/LPS-treated animals, loss of glomerular EC staining correlated with decreased expression of EC-specific transcripts, and impaired kidney function. Selective injury of the glomerular microvasculature with LS toxin conjugate and LPS elicits histologic features of thrombotic microangiopathy and acute kidney failure.

Loss of Apelin exacerbates myocardial infarction adverse remodeling and ischemia-reperfusion injury: therapeutic potential of synthetic Apelin analogues.

BACKGROUND: Coronary artery disease leading to myocardial ischemia is the most common cause of heart failure. Apelin (APLN), the endogenous peptide ligand of the APJ receptor, has emerged as a novel regulator of the cardiovascular system. METHODS AND RESULTS: Here we show a critical role of APLN in myocardial infarction (MI) and ischemia-reperfusion (IR) injury in patients and animal models. Myocardial APLN levels were reduced in patients with ischemic heart failure. Loss of APLN increased MI-related mortality, infarct size, and inflammation with drastic reductions in prosurvival pathways resulting in greater systolic dysfunction and heart failure. APLN deficiency decreased vascular sprouting, impaired sprouting of human endothelial progenitor cells, and compromised in vivo myocardial angiogenesis. Lack of APLN enhanced susceptibility to ischemic injury and compromised functional recovery following ex vivo and in vivo IR injury. We designed and synthesized two novel APLN analogues resistant to angiotensin converting enzyme 2 cleavage and identified one analogue, which mimicked the function of APLN, to be markedly protective against ex vivo and in vivo myocardial IR injury linked to greater activation of survival pathways and promotion of angiogenesis. CONCLUSIONS: APLN is a critical regulator of the myocardial response to infarction and ischemia and pharmacologically targeting this pathway is feasible and represents a new class of potential therapeutic agents.