Novel Function of Nogo-A as Negative Regulator of Endothelial Progenitor Cell Angiogenic Activity: Impact in Oxygen-Induced Retinopathy.

Endothelial Progenitor Cells (EPCs) can actively participate in revascularization in oxygen-induced retinopathy (OIR). Yet the mechanisms responsible for their dysfunction is unclear. Nogo-A, whose function is traditionally related to the inhibition of neurite function in the central nervous system, has recently been documented to display anti-angiogenic pro-repellent properties. Based on the significant impact of EPCs in retinal vascularization, we surmised that Nogo-A affects EPC function, and proceeded to investigate the role of Nogo-A on EPC function in OIR. The expression of Nogo-A and its specific receptor NgR1 was significantly increased in isolated EPCs exposed to hyperoxia, as well as in EPCs isolated from rats subjected to OIR compared with respective controls (EPCs exposed to normoxia). EPCs exposed to hyperoxia displayed reduced migratory and tubulogenic activity, associated with the suppressed expression of prominent EPC-recruitment factors SDF-1/CXCR4. The inhibition of Nogo-A (using a Nogo-66 neutralizing antagonist peptide) or siRNA-NGR1 in hyperoxia-exposed EPCs restored SDF-1/CXCR4 expression and, in turn, rescued the curtailed neovascular functions of EPCs in hyperoxia. The in vivo intraperitoneal injection of engineered EPCs (Nogo-A-inhibited or NgR1-suppressed) in OIR rats at P5 (prior to exposure to hyperoxia) prevented retinal and choroidal vaso-obliteration upon localization adjacent to vasculature; coherently, the inhibition of Nogo-A/NgR1 in EPCs enhanced the expression of key angiogenic factors VEGF, SDF-1, PDGF, and EPO in retina; CXCR4 knock-down abrogated suppressed NgR1 pro-angiogenic effects. The findings revealed that hyperoxia-induced EPC malfunction is mediated to a significant extent by Nogo-A/NgR1 signaling via CXCR4 suppression; the inhibition of Nogo-A in EPCs restores specific angiogenic growth factors in retina and the ensuing vascularization of the retina in an OIR model.

Stimulation of soluble guanylate cyclase activity with riociguat promotes angiogenesis and improves neovascularization after limb ischemia.

BACKGROUND AND AIMS: The NO-cGMP pathway is essential for angiogenesis, vasculogenesis and post-natal neovascularization. The key enzyme responsible for the synthesis of cGMP following binding of NO is soluble guanylate cyclase (sGC). Riociguat is the first member of a novel class of compounds known as sGC stimulators. We tested the hypothesis that stimulation of sGC with riociguat might improve neovascularization in response to ischemia. METHODS: In vitro, the angiogenic effect of riociguat was tested in human umbilical vein endothelial cells (HUVECs). In vivo, neovascularization was investigated in a mouse model of limb ischemia. C57Bl/6 mice were treated by gavage with 3 mg/kg/day of riociguat for a total of 28 days. After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. RESULTS: In a matrigel assay in vitro, riociguat dose-dependently stimulates tubule formation in HUVECs. Cell migration (scratch assay) is also increased in HUVECs treated with riociguat. At the molecular level, riociguat treatment leads to rapid activation of the p44/p42 MAP kinase pathway in HUVECs. Inhibition of protein kinase G (PKG) activity supresses both p44/p42 MAP kinase activation and angiogenesis in HUVECs treated with riociguat. In vivo, treatment with riociguat improves blood flow recovery after ischemia (Laser Doppler imaging), and increases capillary density in ischemic muscles (CD31 immunostaining). Clinically, this is associated with a significant decrease of ambulatory impairment and ischemic damages. Interestingly, mice treated with riociguat also show a 94% increase in the number of bone marrow-derived pro-angiogenic cells (PACs) compared to control mice. Moreover, riociguat treatment is associated with a significant improvement of PAC functions including migratory capacity, adhesion to an endothelial monolayer, and integration into endothelial tubular networks. CONCLUSIONS: The sGC stimulator riociguat promotes angiogenesis and improves neovascularization after ischemia. The mechanism involves PKG-dependent activation of p44/p42 MAP kinase pathway, together with an improvement of PAC number and functions. sGC stimulation could constitute a novel therapeutic strategy to reduce tissue ischemia in patients with severe atherosclerotic diseases.

Tyrosine-Protein Phosphatase Non-receptor Type 9 (PTPN9) Negatively Regulates the Paracrine Vasoprotective Activity of Bone-Marrow Derived Pro-angiogenic Cells: Impact on Vascular Degeneration in Oxygen-Induced Retinopathy.

BACKGROUND AND AIM: Insufficient post-ischemic neovascularization is an initial key step in the pathogenesis of Oxygen-Induced Retinopathy (OIR). During neovascularization, pro-angiogenic cells (PACs) are mobilized from the bone marrow and integrate into ischemic tissues to promote angiogenesis. However, the modulation of PAC paracrine activity during OIR and the specific mechanisms involved remain to be explored. Because Tyrosine-protein phosphatase non-receptor type 9 (PTPN9) is reported to be a negative regulator of stem cell differentiation and angiogenesis signaling, we investigated its effect on PAC activity in the context of OIR. METHODS AND RESULTS: In a rat model of OIR, higher levels of PTPN9 in the retina and in bone marrow derived PACs are associated with retinal avascular areas, lower levels of the mobilization factor SDF-1 and decreased number of CD34+/CD117+/CD133+ PACs. PACs exposed ex vivo to hyperoxia display increased PTPN9 expression, which is associated with impaired ability of PAC secretome to promote angiogenesis ex vivo (choroidal vascular sprouting) and in vitro (endothelial cell tubule formation) compared to the secretome of PACs maintained in normoxia. Suppression of PTPN9 (using siRNA) increases VEGF and SDF-1 expression to normalize PAC secretome during hyperoxia, leading to restored angiogenic ability of PAC secretome. Moreover, endothelial cells exposed to the secretome of siPTPN9-treated PACs expressed increased levels of activated form of VEGF receptor 2 (VEGFR2). In the rat model of OIR, intravitreal injection of secretome from siPTPN9-treated PACs significantly reduced retinal vaso-obliteration; this was associated with higher retinal levels of VEGF/SDF-1, and increased recruitment of PACs (CD34+ cells) to the retinal and choroidal vessels. CONCLUSION: Our results suggest that hyperoxia alters the paracrine proangiogenic activity of BM-PACs by inducing PTPN9, which can contribute to impair post-ischemic revascularization in the context of OIR. Targeting PTPN9 restores PAC angiogenic properties, and provide a new target for vessel integrity in ischemic retinopathies.

Nutraceutical Targeting of Inflammation-Modulating microRNAs in Severe Forms of COVID-19: A Novel Approach to Prevent the Cytokine Storm.

The coronavirus disease 2019 (COVID-19) pandemic has become the number one health problem worldwide. As of August 2020, it has affected more than 18 million humans and caused over 700,000 deaths worldwide. COVID-19 is an infectious disease that can lead to severe acute respiratory syndrome. Under certain circumstances, the viral infection leads to excessive and uncontrolled inflammatory response, which is associated with the massive release of inflammatory cytokines in pulmonary alveolar structures. This phenomenon has been referred to as the "cytokine storm," and it is closely linked to lung injury, acute respiratory syndrome and mortality. Unfortunately, there is currently no vaccine available to prevent the infection, and no effective treatment is available to reduce the mortality associated with the severe form of the disease. The cytokine storm associate with COVID-19 shows similarities with those observed in other pathologies such as sepsis, acute respiratory distress syndrome, acute lung injury and other viral infection including severe cases of influenza. However, the specific mechanisms that cause and modulate the cytokine storm in the different conditions remain to be determined. micro-RNAs are important regulators of gene expression, including key inflammatory cytokines involved in the massive recruitment of immune cells to the lungs such as IL1ß, IL6, and TNFα. In recent years, it has been shown that nutraceutical agents can modulate the expression of miRs involved in the regulation of cytokines in various inflammatory diseases. Here we review the potential role of inflammatory-regulating-miRs in the cytokine storm associated with COVID-19, and propose that nutraceutical agents may represent a supportive therapeutic approach to modulate dysregulated miRs in this condition, providing benefits in severe respiratory diseases.

An allosteric interleukin-1 receptor modulator mitigates inflammation and photoreceptor toxicity in a model of retinal degeneration.

BACKGROUND: Inflammation and particularly interleukin-1ß (IL-1ß), a pro-inflammatory cytokine highly secreted by activated immune cells during early AMD pathological events, contribute significantly to retinal neurodegeneration. Here, we identify specific cell types that generate IL-1ß and harbor the IL-1 receptor (IL-1R) and pharmacologically validate IL-1ß's contribution to neuro-retinal degeneration using the IL-1R allosteric modulator composed of the amino acid sequence rytvela (as well as the orthosteric antagonist, Kineret) in a model of blue light-induced retinal degeneration. METHODS: Mice were exposed to blue light for 6 h and sacrificed 3 days later. Mice were intraperitoneally injected with rytvela, Kineret, or vehicle twice daily for 3 days. The inflammatory markers F4/80, NLRP3, caspase-1, and IL-1ß were assessed in the retinas. Single-cell RNA sequencing was used to determine the cell-specific expression patterns of retinal Il1b and Il1r1. Macrophage-induced photoreceptor death was assessed ex vivo using retinal explants co-cultured with LPS-activated bone marrow-derived macrophages. Photoreceptor cell death was evaluated by the TUNEL assay. Retinal function was assessed by flash electroretinography. RESULTS: Blue light markedly increased the mononuclear phagocyte recruitment and levels of inflammatory markers associated with photoreceptor death. Co-localization of NLRP3, caspase-1, and IL-1ß with F4/80+ mononuclear phagocytes was clearly detected in the subretinal space, suggesting that these inflammatory cells are the main source of IL-1ß. Single-cell RNA sequencing confirmed the immune-specific expression of Il1b and notably perivascular macrophages in light-challenged mice, while Il1r1 expression was found primarily in astrocytes, bipolar, and vascular cells. Retinal explants co-cultured with LPS/ATP-activated bone marrow-derived macrophages displayed a high number of TUNEL-positive photoreceptors, which was abrogated by rytvela treatment. IL-1R antagonism significantly mitigated the inflammatory response triggered in vivo by blue light exposure, and rytvela was superior to Kineret in preserving photoreceptor density and retinal function. CONCLUSION: These findings substantiate the importance of IL-1ß in neuro-retinal degeneration and revealed specific sources of Il1b from perivascular MPs, with its receptor Ilr1 being separately expressed on surrounding neuro-vascular and astroglial cells. They also validate the efficacy of rytvela-induced IL-1R modulation in suppressing detrimental inflammatory responses and preserving photoreceptor density and function in these conditions, reinforcing the rationale for clinical translation.

MicroRNA Expression Profiling of Bone Marrow-Derived Proangiogenic Cells (PACs) in a Mouse Model of Hindlimb Ischemia: Modulation by Classical Cardiovascular Risk Factors.

BACKGROUND: Classical cardiovascular risk factors (CRFs) are associated with impaired angiogenic activities of bone marrow-derived proangiogenic cells (PACs) related to peripheral artery diseases (PADs) and ischemia-induced neovascularization. MicroRNAs (miRs) are key regulators of gene expression, and they are involved in the modulation of PAC function and PAC paracrine activity. However, the effects of CRFs on the modulation of miR expression in PACs are unknown. AIMS AND METHODS: We used a model of hindlimb ischemia and next-generation sequencing to perform a complete profiling of miRs in PACs isolated from the bone marrow of mice subjected to three models of CRFs: aging, smoking (SMK) and hypercholesterolemia (HC). RESULTS: Approximately 570 miRs were detected in PACs in the different CRF models. When excluding miRs with a very low expression level (<100 RPM), 40 to 61 miRs were found to be significantly modulated by aging, SMK, or HC. In each CRF condition, we identified downregulated proangiogenic miRs and upregulated antiangiogenic miRs that could contribute to explain PAC dysfunction. Interestingly, several miRs were similarly downregulated (e.g., miR-542-3p, miR-29) or upregulated (e.g., miR-501, miR-92a) in all CRF conditions. In silico approaches including Kyoto Encyclopedia of Genes and Genomes and cluster dendogram analyses identified predictive effects of these miRs on pathways having key roles in the modulation of angiogenesis and PAC function, including vascular endothelial growth factor signaling, extracellular matrix remodeling, PI3K/AKT/MAPK signaling, transforming growth factor beta (TGFb) pathway, p53, and cell cycle progression. CONCLUSION: This study describes for the first time the effects of CRFs on the modulation of miR profile in PACs related to PAD and ischemia-induced neovascularization. We found that several angiogenesis-modulating miRs are similarly altered in different CRF conditions. Our findings constitute a solid framework for the identification of miRs that could be targeted in PACs in order to improve their angiogenic function and for the future development of novel therapies to improve neovascularization and reduce tissue damage in patients with severe PAD.

Reduced expression of microRNA-130a promotes endothelial cell senescence and age-dependent impairment of neovascularization.

Aging is associated with impaired neovascularization in response to ischemia. MicroRNAs are small noncoding RNAs emerging as key regulators of physiological and pathological processes. Here we investigated the potential role of microRNAs in endothelial cell senescence and age-dependent impairment of neovascularization. Next generation sequencing and qRT-PCR analyses identified miR-130a as a pro-angiogenic microRNA which expression is significantly reduced in old mouse aortic endothelial cells (ECs). Transfection of young ECs with a miR-130a inhibitor leads to accelerated senescence and reduced angiogenic functions. Conversely, forced expression of miR-130a in old ECs reduces senescence and improves angiogenesis. In a mouse model of hindlimb ischemia, intramuscular injection of miR-130a mimic in older mice restores blood flow recovery and vascular densities in ischemic muscles, improves mobility and reduces tissue damage. miR-130a directly targets antiangiogenic homeobox genes MEOX2 and HOXA5. MEOX2 and HOXA5 are significantly increased in the ischemic muscles of aging mice, but forced expression of miR-130a reduces the expression of these factors. miR-130a treatment after ischemia is also associated with increased number and improved functional activities of pro-angiogenic cells (PACs). Forced expression of miR-130a could constitute a novel strategy to improve blood flow recovery and reduce ischemia in older patients with ischemic vascular diseases.

Novel Anti-Interleukin-1ß Therapy Preserves Retinal Integrity: A Longitudinal Investigation Using OCT Imaging and Automated Retinal Segmentation in Small Rodents.

Retinopathy of prematurity (ROP) is the leading cause of blindness in neonates. Inflammation, in particular interleukin-1ß (IL-1ß), is increased in early stages of the disorder, and contributes to inner and outer retinal vasoobliteration in the oxygen-induced retinopathy (OIR) model of ROP. A small peptide antagonist of IL-1 receptor, composed of the amino acid sequence, rytvela, has been shown to exert beneficial anti-inflammatory effects without compromising immunovigilance-related NF-κB in reproductive tissues. We conducted a longitudinal study to determine the efficacy of "rytvela" in preserving the integrity of the retina in OIR model, using optical coherence tomography (OCT) which provides high-resolution cross-sectional imaging of ocular structures in vivo. Sprague-Dawley rats subjected to OIR and treated or not with "rytvela" were compared to IL-1 receptor antagonist (Kineret). OCT imaging and custom automated segmentation algorithm used to measure retinal thickness (RT) were obtained at P14 and P30; gold-standard immunohistochemistry (IHC) was used to confirm retinal anatomical changes. OCT revealed significant retinal thinning in untreated animals by P30, confirmed by IHC; these changes were coherently associated with increased apoptosis. Both rytvela and Kineret subsided apoptosis and preserved RT. As anticipated, Kineret diminished both SAPK/JNK and NF-κB axes, whereas rytvela selectively abated the former which resulted in preserved monocyte phagocytic function. Altogether, OCT imaging with automated segmentation is a reliable non-invasive approach to study longitudinally retinal pathology in small animal models of retinopathy.

MicroRNA-96 Promotes Vascular Repair in Oxygen-Induced Retinopathy-A Novel Uncovered Vasoprotective Function.

BACKGROUND AND AIMS: Vascular degeneration is a hallmark in the pathogenesis of oxygen-induced retinopathy (OIR). Dysregulation of microRNAs (miRNAs), key regulators of genes expressions, has been implicated in the regulation of ocular angiogenesis. However, miRNAs specific functions in impaired vascular development during OIR are poorly understood. Herein, we identified miR-96 as one of the most highly expressed miRNAs in the retina and choroid during vascular development and investigated the potential role of miR-96 on microvascular degeneration in a rat OIR model. METHODS AND RESULTS: Next generation sequencing (NGS) and qRT-PCR analysis showed that miR-96 maintain high levels of expression during ocular vascular development. Nevertheless, miR-96 was significantly downregulated in the retina and choroid of OIR rats (80% O2 from P5 to P10) during the phase of microvascular degeneration. Similarly, human retinal microvascular endothelial cells (HRMEC) subjected to hyperoxia (80% O2) showed a significant downregulation of miR-96 evaluated by qPCR. Interestingly, HRMEC supplemented with miR-96 regulated positively the expression of several key angiogenic factors including VEGF and ANG-2. To explore the angiogenic activity of miR-96 on HRMEC, we performed a gain/loss of function study. In a similar way to hyperoxia exposure, we observed a robust angiogenic impairment (tubulogenesis and migration) on HRMEC transfected with an antagomiR-96. Conversely, overexpression of miR-96 stimulated the angiogenic activity of HRMEC and protected against hyperoxia-induced endothelial dysfunction. Finally, we evaluated the potential vasoprotective function of miR-96 in OIR animals. Rat pups intravitreally supplemented with miR-96 mimic (1 mg/kg) displayed a significant preservation of retinal/choroidal microvessels at P10 compared to controls. This result was consistent with the maintenance of physiologic levels of VEGF and ANG-2 in the OIR retina. CONCLUSION: This study demonstrates that miR-96 regulates the expression of angiogenic factors (VEGF/ANG-2) associated to the maintenance of retinal and choroidal microvasculature during physiological and pathological conditions. Intravitreal supplementation of miR-96 mimic could constitute a novel therapeutic strategy to improve vascular repair in OIR and other ischemic retinopathies.

Propranolol Attenuates Proangiogenic Activity of Mononuclear Phagocytes: Implication in Choroidal Neovascularization.

Purpose: Targeting ß-adrenergic receptor signaling with propranolol has emerged as a potential candidate to counteract choroidal neovascularization (CNV). Little is known of its effect on macrophages, which play a critical role in CNV. We investigated the effect of propranolol on angiogenic response of mononuclear phagocytes (MPs). Methods: The angiogenic effect of propranolol was evaluated in laser-induced CNV model. Mice received intraperitoneal injections of propranolol (6 mg/kg/d) or vehicle. CNV area and inflammatory cells were determined respectively by using lectin staining and an anti-IBA-1 antibody on RPE/choroid flat mounts. Inflammatory gene expression was evaluated by quantitative (q) PCR analysis. Mechanisms of propranolol was studied in MP cell lines J774 and RAW264.7 and in primary peritoneal macrophages. Expression of pro- and antiangiogenic mediators was studied. In addition, effects of propranolol treatment of MPs was assessed on choroidal explant. Results: CNV was attenuated by propranolol and concomitantly associated with decreased inflammatory mediators IL-6 and TNFα, albeit with accumulation of (ß-adrenoceptor harboring) MPs in the CNV area. Conditioned media from MPs preincubated with propranolol exerted antiangiogenic effects. Treatment of J774 confirmed the attenuation of inflammatory response to propranolol and increased cleaved caspase-3 on choroidal explant. We found that propranolol increased pigment epithelium-derived factor (PEDF) expression in MPs. Trapping of PEDF with an antibody abrogated antiangiogenic effects of propranolol. PEDF was also detected in CNV-associated MPs. Conclusions: We hereby show that propranolol confers on MPs antiangiogenic properties by increasing PEDF expression, which complements its effects on vascular tissue resulting in inhibition of choroidal vasoproliferation in inflammatory conditions. The study supports possible use of propranolol as a therapeutic modality for CNV.

Forced expression of microRNA-146b reduces TRAF6-dependent inflammation and improves ischemia-induced neovascularization in hypercholesterolemic conditions.

BACKGROUND AND AIMS: MicroRNA (miR)-146 is a key regulator of inflammation, endothelial activation and atherosclerosis. This study sought to define its potential role for the modulation of ischemia-induced neovascularization in atherosclerotic conditions. METHODS: Next generation sequencing and qRT-PCR analyses were used to compare microRNA expression in the ischemic muscles of hypercholesterolemic ApoE-deficient (ApoE-/-) mice vs. wild type mice, and in HUVECs exposed or not to oxLDL. Neovascularization was investigated in a mouse model of hindlimb ischemia and the functional activities of HUVECs and pro-angiogenic cells (PACs) were assessed in vitro. RESULTS: We found that miR-146b (but not miR-146a) is significantly reduced in the ischemic muscles of ApoE-/- mice, and in HUVECs exposed to oxLDL. Inhibition of miR-146b reduces angiogenesis in vitro, whereas forced expression of miR-146b rescues oxLDL-mediated impairment of endothelial cell proliferation and tube formation. Mechanistically, miR146b directly targets tumor necrosis factor-alpha (TNFa) Receptor Associated Factor 6 (TRAF6) to inhibit inflammation. We found that hypercholesterolemia and oxLDL exposure are associated with higher levels of TRAF6, and increased expression of TNFa. However, forced expression of miR-146b in high cholesterol conditions reduces the expression of these inflammatory factors. In vivo, intramuscular injection of miR-146b mimic reduces ischemic damages and restores blood flow recuperation and capillary density in the ischemic muscles of ApoE-/- mice. Treatment with miR-146b also increases the number and functional activities of pro-angiogenic cells (PACs). CONCLUSIONS: Hypercholesterolemia is associated with reduced expression of miR-146b, which increases TRAF6-dependent inflammation and is associated with poor neovascularization in response to ischemia. Forced expression of miR-146b using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.

The Inability of the Choroid to Revascularize in Oxygen-Induced Retinopathy Results from Increased p53/miR-Let-7b Activity.

Retinopathy of prematurity (ROP) is characterized by an initial retinal avascularization, followed by pathologic neovascularization. Recently, choroidal thinning has also been detected in children formerly diagnosed with ROP; a similar sustained choroidal thinning is observed in ROP models. But the mechanism underlying the lack of choroidal revascularization remains unclear and was investigated in an oxygen-induced retinopathy (OIR) model. In OIR, evidence of senescence was detected, preceded by oxidative stress in the choroid and the retinal pigment epithelium. This was associated with a global reduction of proangiogenic factors, including insulin-like growth factor 1 receptor (Igf1R). Coincidentally, tumor suppressor p53 was highly expressed in the OIR retinae. Curtailing p53 activity resulted in reversal of senescence, normalization of Igf1r expression, and preservation of choroidal integrity. OIR-induced down-regulation of Igf1r was mediated at least partly by miR-let-7b as i) let-7b expression was augmented throughout and beyond the period of oxygen exposure, ii) let-7b directly targeted Igf1r mRNA, and iii) p53 knock-down blunted let-7b expression, restored Igf1r expression, and elicited choroidal revascularization. Finally, restoration of Igf1r expression rescued choroid thickness. Altogether, this study uncovers a significant mechanism for defective choroidal revascularization in OIR, revealing a new role for p53/let-7b/IGF-1R axis in the retina. Future investigations on this (and connected) pathway could further our understanding of other degenerative choroidopathies, such as geographic atrophy.

MicroRNA expression profile in retina and choroid in oxygen-induced retinopathy model.

BACKGROUND: Ischemic retinopathies (IRs) are leading causes of visual impairment. They are characterized by an initial phase of microvascular degeneration and a second phase of aberrant pre-retinal neovascularization (NV). microRNAs (miRNAs) regulate gene expression, and a number play a role in normal and pathological NV. But, post-transcriptional modulation of miRNAs in the eye during the development of IRs has not been systematically evaluated. AIMS & METHODS: Using Next Generation Sequencing (NGS) we profiled miRNA expression in the retina and choroid during vasodegenerative and NV phases of oxygen-induced retinopathy (OIR). RESULTS: Approximately 20% of total miRNAs exhibited altered expression (up- or down-regulation); 6% of miRNA were found highly expressed in retina and choroid of rats subjected to OIR. During OIR-induced vessel degeneration phase, miR-199a-3p, -199a-5p, -1b, -126a-3p displayed a robust decreased expression (> 85%) in the retina. While in the choroid, miR-152-3p, -142-3p, -148a-3p, -532-3p were upregulated (>200%) and miR-96-5p, -124-3p, -9a-3p, -190b-5p, -181a-1-3p, -9a-5p, -183-5p were downregulated (>70%) compared to controls. During peak pathological NV, miR-30a-5p, -30e-5p and 190b-5p were markedly reduced (>70%), and miR-30e-3p, miR-335, -30b-5p strongly augmented (by up to 300%) in the retina. Whereas in choroid, miR-let-7f-5p, miR-126a-5p and miR-101a-3p were downregulated by (>81%), and miR-125a-5p, let-7e-5p and let-7g-5p were upregulated by (>570%) during NV. Changes in miRNA observed using NGS were validated using qRT-PCR for the 24 most modulated miRNAs. In silico approach to predict miRNA target genes (using algorithms of miRSystem database) identified potential new target genes with pro-inflammatory, apoptotic and angiogenic properties. CONCLUSION: The present study is the first comprehensive description of retinal/choroidal miRNAs profiling in OIR (using NGS technology). Our results provide a valuable framework for the characterization and possible therapeutic potential of specific miRNAs involved in ocular IR-triggered inflammation, angiogenesis and degeneration.

Dual functions of ARP101 in targeting membrane type-1 matrix metalloproteinase: Impact on U87 glioblastoma cell invasion and autophagy signaling.

Membrane type-1 matrix metalloproteinase (MT1-MMP) possesses both extracellular proteolytic and intracellular signal-transducing functions in tumorigenesis. An imbalance in MT1-MMP expression and/or function triggers a metastatic, invasive, and therapy resistance phenotype. MT1-MMP is involved in extracellular matrix (ECM) proteolysis, activation of latent MMPs, as well as in autophagy signaling in human hepatoma and glioblastoma cells. A low autophagy index in tumorigenesis has been inferred by recent studies where autophagic capacity was decreased during tumor progression. Here, we establish ARP101 as a dual-function small-molecule inhibitor against MT1-MMP ECM hydrolysis and autophagy signal-transducing functions in a model of grade IV glioblastoma cells. ARP101 inhibited concanavalin-A-mediated proMMP-2 activation into MMP-2, as well as MT1-MMP auto-proteolytic processing. When overexpressing recombinant Wt MT1-MMP, ARP101 inhibited proMMP-2 activation and triggered the formation of MT1-MMP oligomers that required trafficking to the plasma membrane. ARP101 further induced cell autophagy as reflected by increased formation of acidic vacuole organelles, LC3 puncta, and autophagy-related protein ATG9 transcription. These were all significantly reversed upon siRNA-mediated gene silencing of MT1-MMP. ARP101 can thus concomitantly inhibit MT1-MMP extracellular catalytic function and exploit its intracellular transducing signal function to trigger autophagy-mediated cell death in U87 glioblastoma cancer cells.

Neonatal exposure to high oxygen levels leads to impaired ischemia-induced neovascularization in adulthood.

Adverse perinatal conditions can lead to developmental programming of cardiovascular diseases. Prematurely born infants are often exposed to high oxygen levels, which in animal models has been associated with endothelial dysfunction, hypertension, and cardiac remodeling during adulthood. Here we found that adult mice that have been transiently exposed to O2 after birth show defective neovasculariation after hindlimb ischemia, as demonstrated by impaired blood flow recovery, reduced vascular density in ischemic muscles and increased tissue damages. Ischemic muscles isolated from mice exposed to O2 after birth exhibit increased oxidative stress levels and reduced expression of superoxide dismutase 1 (SOD1) and vascular endothelial growth factor (VEGF). Pro-angiogenic cells (PACs) have been shown to have an important role for postnatal neovascularisation. We found that neonatal exposure to O2 is associated with reduced number of PACs in adults. Moreover, the angiogenic activities of both PACs and mature mouse aortic endothelial cells (MAECs) are significantly impaired in mice exposed to hyperoxia after birth. Our results indicate that neonatal exposure to high oxygen levels leads to impaired ischemia-induced neovascularization during adulthood. The mechanism involves deleterious effects on oxidative stress levels and angiogenic signals in ischemic muscles, together with dysfunctional activities of PACs and mature endothelial cells.

MicroRNA-150 Modulates Ischemia-Induced Neovascularization in Atherosclerotic Conditions.

OBJECTIVE: Hypercholesterolemia is an atherosclerotic condition that is associated with impaired neovascularization in response to ischemia. This study sought to define the role of microRNAs in that pathophysiology. APPROACH AND RESULTS: Next-generation sequencing and quantitative reverse transcription polymerase chain reaction analyses identified miR-150 as a proangiogenic microRNA, which expression is significantly reduced in the ischemic muscles of hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice, and in human umbilical vein endothelial cells exposed to oxidized low-density lipoprotein. Forced expression of miR-150 using a miR mimic could rescue oxidized low-density lipoprotein-mediated impairment of endothelial cell migration and tubule formation in vitro. In a mouse model of hindlimb ischemia, intramuscular injection of miR-150 mimic restored blood flow recuperation, vascular densities in ischemic muscles, and functional mobility in ApoE-/- mice. Treatment of ApoE-/- mice with miR-150 also increased the number and the activities of proangiogenic cells. miR-150 targets SRC kinase signaling inhibitor 1, an important regulator of Src (proto-oncogene tyrosine-protein kinase Src) activity. Here we found that hypercholesterolemia and oxidized low-density lipoprotein exposure are associated with increased SRC kinase signaling inhibitor 1 expression and decreased Src activity. However, treatment with miR-150 mimic reduces SRC kinase signaling inhibitor 1 expression and restores Src and downstream endothelial nitric oxide synthase and Akt (protein kinase B) activities both in vitro and in vivo. We also demonstrate the interrelation between miR-150 and SRC kinase signaling inhibitor 1 and their importance for endothelial cell angiogenic activities. CONCLUSIONS: Hypercholesterolemia is associated with reduced expression of miR-150, impaired Src signaling, and inefficient neovascularization in response to ischemia. Forced expression of miR-150 using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.

Reduced expression of let-7f activates TGF-ß/ALK5 pathway and leads to impaired ischaemia-induced neovascularization after cigarette smoke exposure.

This study sought to determine the potential role of microRNAs (miRNAs) in the detrimental effects of cigarette smoke on angiogenesis and neovascularization. Using large-scale miRNA profiling and qRT-PCR analyses, we identified let-7f as a pro-angiogenic miRNA which expression is significantly reduced in HUVECs treated with cigarette smoke extracts (CSE), and in the ischemic muscles of mice that are exposed to cigarette smoke (MES). In a mouse model of hindlimb ischaemia, intramuscular injection of let-7f mimic restored ischaemia-induced neovascularization in MES. Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let-7f mimic. Clinically, this was associated with reduced ambulatory impairment and hindlimb ischaemic damage. Treatment with let-7f mimic could also rescue pro-angiogenic cell (PAC) number and function (attachment, proliferation, migration) in MES. ALK5 (TGF-ßR1), an important modulator of angiogenesis, is a target of let-7f. Here we show that ALK5 is increased in HUVECs exposed to CSE and in the ischaemic muscles of MES. This is associated with a downstream activation of the anti-angiogenic factors SMAD2/3 and PAI-1. Importantly, treatment with let-7f mimic reduces the expression of ALK5, SMAD2/3 and PAI-1 both in vitro and in vivo. Moreover, let-7f overexpression or ALK5 inhibition can rescue angiogenesis in HUVECs exposed to CSE. Cigarette smoke exposure is associated with reduced expression of let-7f and activation of the anti-angiogenic TGF-ß/ALK5 pathway. Overexpression of let-7f using a miRNA mimic could constitute a novel therapeutic strategy to improve ischaemia-induced neovascularization in pathological conditions.

Direct renin inhibition with aliskiren improves ischemia-induced neovascularization: blood pressure-independent effect.

BACKGROUND: Renin is the rate limiting step for the activation of the renin-angiotensin-aldosterone system, which is linked to the development of endothelial dysfunction, hypertension and atherosclerosis. However, the specific role of renin during physiological responses to tissue ischemia is currently unknown. Aliskiren is the only direct renin inhibitor that is clinically used as an orally active antihypertensive drug. Here we tested the hypothesis that aliskiren might improve neovascularization in response to ischemia. METHODS AND RESULTS: At a dose that did not modulate blood pressure (10 mg/kg), aliskiren led to improved blood flow recovery after hindlimb ischemia in C57BL/6 mice (Doppler flow ratios 0.71 ± 0.07 vs. 0.55 ± 0.03; P < 0.05). In ischemic muscles, treatment with aliskiren was associated with a significant increase of vascular density, reduced oxidative stress levels and increased expression of VEGF and eNOS. Aliskiren treatment also significantly increased the number of bone marrow-derived endothelial progenitor cells (EPCs) after hindlimb ischemia. Moreover, the angiogenic properties of EPCs (migration, adhesion, integration into tubules) were significantly improved in mice treated with aliskiren. In vitro, aliskiren improves cellular migration and tubule formation in HUVECs. This is associated with an increased expression of nitric oxide (NO), and a significant reduction of oxidative stress levels. Importantly, the angiogenic properties of aliskiren in vitro and in vivo are completely abolished following treatment with the NOS inhibitor l-NAME. CONCLUSION: Direct renin inhibition with aliskiren leads to improved ischemia-induced neovascularization that is not dependant on blood pressure lowering. The mechanism involves beneficial effects of aliskiren on oxidative stress and NO angiogenic pathway, together with an increase in the number and the functional activities of EPCs.

Psychological stress impairs ischemia-induced neovascularization: Protective effect of fluoxetine.

BACKGROUND: Psychological stress (PS) has been associated with the development of cardiovascular diseases and adverse long-term outcomes after ischemic events. However, the precise mechanisms involved are not completely understood. Here we investigated the effect of PS on ischemia-induced neovascularization, and the potential therapeutic effect of fluoxetine in this condition. METHODS AND RESULTS: Balb/c mice were subjected or not to chronic restraint stress. After 3 weeks, hindlimb ischemia was surgically induced by femoral artery removal. We found that blood flow recovery was significantly impaired in mice exposed to PS compared to controls (Doppler flow ratio (DFR) 0.61 ± 0.07 vs. 0.80 ± 0.07, p < 0.05). At the microvascular level, capillary density was significantly reduced in ischemic muscles of mice exposed to PS (38 ± 1 vs. 74 ± 3 capillaries per field, p < 0.001). This correlated with increased oxidative stress levels and reduced expression of VEGF and VEGF signalling molecules (p44/p42 MAPK, Akt) in ischemic muscles. We found that the number of pro-angiogenic cells (PACs) was significantly reduced in mice exposed to PS. In addition, oxidative stress levels (DCF-DA, DHE) were increased in PACs isolated from mice exposed to PS, and this was associated with impaired PAC functional activities (migration, adhesion, and integration into tubules). Importantly, treatment of mice exposed to PS with the selective serotonin reuptake inhibitor (SSRI) fluoxetine improved all the angiogenic parameters, and completely rescued PS-induced impairment of neovascularization. CONCLUSION: PS impairs ischemia-induced neovascularization. Potential mechanisms involved include reduced activation of the VEGF pathway in ischemic tissues, increased oxidative stress levels and reduced number and functional activities of PACs. Our results suggest that fluoxetine may represent a novel therapeutic strategy to improve neovascularization and reduce ischemia in patients suffering from cardiovascular diseases and exposed to PS.

Elsibucol inhibits atherosclerosis following arterial injury: multifunctional effects on cholesterol levels, oxidative stress and inflammation.

BACKGROUND: Elsibucol is a metabolically stable derivative of probucol with antioxidant, anti-inflammatory and antiproliferative properties. Here we investigated the effect of elsibucol on the development of atherosclerosis following arterial injury in hypercholesterolemic rabbits. METHODS AND RESULTS: New Zealand White rabbits were fed a high cholesterol diet that was supplemented or not with 0.5% elsibucol, 1% elsibucol or 1% probucol. An angioplasty of the iliac artery was performed after 3 weeks of diet. We found that treatment with elsibucol significantly decreases blood total cholesterol, LDLc and triglyceride levels. This is associated with a significant 46% reduction of neointimal hyperplasia following arterial injury. Interestingly, the effect of elsibucol on cholesterol levels and neointimal formation appears to be more pronounced than that of probucol. In vitro, elsibucol reduces vascular smooth muscle cell proliferation without affecting cell viability. In vivo, treatment with elsibucol is associated with a significant reduction of cellular proliferation (PCNA immunostaining), oxidative stress (nitrotyrosine immunostaining), VCAM-1 expression and macrophage infiltration in injured arteries. Despite its potent effect on neointimal hyperplasia, elsibucol does not prevent endothelial healing (Evans blue staining) following arterial injury. CONCLUSIONS: In hypercholesterolemic animals, elsibucol inhibits atherosclerosis and preserves endothelial healing following arterial injury. The mechanisms involved include lowering of blood cholesterol levels together with a reduction of oxidative stress and inflammation in injured arteries.