Evaluation of interleukin-1 and interleukin-6 receptor antagonists in a murine model of acute lung injury.

The acute exudative phase of acute respiratory distress syndrome (ARDS), a severe form of respiratory failure, is characterized by alveolar damage, pulmonary oedema, and an exacerbated inflammatory response. There is no effective treatment for this condition, but based on the major contribution of inflammation, anti-inflammatory strategies have been evaluated in animal models and clinical trials, with conflicting results. In COVID-19 ARDS patients, interleukin (IL)-1 and IL-6 receptor antagonists (IL-1Ra and IL-6Ra, kineret and tocilizumab, respectively) have shown some efficacy. Moreover, we have previously developed novel peptides modulating IL-1R and IL-6R activity (rytvela and HSJ633, respectively) while preserving immune vigilance and cytoprotective pathways. We aimed to assess the efficacy of these novel IL-1Ra and IL-6Ra, compared to commercially available drugs (kineret, tocilizumab) during the exudative phase (day 7) of bleomycin-induced acute lung injury (ALI) in mice. Our results first showed that none of the IL-1Ra and IL-6Ra compounds attenuated bleomycin-induced weight loss and venous P C O 2 ${P_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ increase. Histological analyses and lung water content measurements also showed that these drugs did not improve lung injury scores or pulmonary oedema, after the bleomycin challenge. Finally, IL-1Ra and IL-6Ra failed to alleviate the inflammatory status of the mice, as indicated by cytokine levels and alveolar neutrophil infiltration. Altogether, these results indicate a lack of beneficial effects of IL-1R and IL-6R antagonists on key parameters of ALI in the bleomycin mouse model.

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.

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.

Tetrahydrobiopterin (BH4) deficiency is associated with augmented inflammation and microvascular degeneration in the retina.

BACKGROUND: Tetrahydrobiopterin (BH4) is an essential cofactor in multiple metabolic processes and plays an essential role in maintaining the inflammatory and neurovascular homeostasis. In this study, we have investigated the deleterious effects of BH4 deficiency on retinal vasculature during development. METHODS: hph-1 mice, which display deficiency in BH4 synthesis, were used to characterize the inflammatory effects and the integrity of retinal microvasculature. BH4 levels in retinas from hph-1 and wild type (WT) mice were measured by LC-MS/MS. Retinal microvascular area and microglial cells number were quantified in hph-1 and WT mice at different ages. Retinal expression of pro-inflammatory, anti-angiogenic, and neuronal-derived factors was analyzed by qPCR. BH4 supplementation was evaluated in vitro, ex-vivo, and in vivo models. RESULTS: Our findings demonstrated that BH4 levels in the retina from hph-1 mice were significantly lower by ~ 90% at all ages analyzed compared to WT mice. Juvenile hph-1 mice showed iris atrophy, persistent fetal vasculature, significant increase in the number of microglial cells (p < 0.01), as well as a marked degeneration of the retinal microvasculature. Retinal microvascular alterations in juvenile hph-1 mice were associated with a decreased expression in Norrin (0.2-fold) and its receptor Frizzled-4 (FZD4; 0.51-fold), as well as with an augmented expression of pro-inflammatory factors such as IL-6 (3.2-fold), NRLP-3 (4.4-fold), IL-1ß (8.6-fold), and the anti-angiogenic factor thrombospondin-1 (TSP-1; 17.5-fold). We found that TSP-1 derived from activated microglial cells is a factor responsible of inducing microvascular degeneration, but BH4 supplementation markedly prevented hyperoxia-induced microglial activation in vitro and microvascular injury in an ex-vivo model of microvascular angiogenesis and an in vivo model of oxygen-induced retinopathy (OIR). CONCLUSION: Our findings reveal that BH4 is a key cofactor in regulating the expression of inflammatory and anti-angiogenic factors that play an important function in the maintenance of retinal microvasculature.

Choroidal Involution Is Associated with a Progressive Degeneration of the Outer Retinal Function in a Model of Retinopathy of Prematurity: Early Role for IL-1ß.

Retinopathy of prematurity (ROP), the most common cause of blindness in premature infants, has long been associated with inner retinal alterations. However, recent studies reveal outer retinal dysfunctions in patients formerly afflicted with ROP. We have recently demonstrated that choroidal involution occurs early in retinopathy. Herein, we investigated the mechanisms underlying the choroidal involution and its long-term impact on retinal function. An oxygen-induced retinopathy (OIR) model was used. In vitro and ex vivo assays were applied to evaluate cytotoxic effects of IL-1ß on choroidal endothelium. Electroretinogram was used to evaluate visual function. We found that proinflammatory IL-1ß was markedly increased in retinal pigment epithelium (RPE)/choroid and positively correlated with choroidal degeneration in the early stages of retinopathy. IL-1ß was found to be cytotoxic to choroid in vitro, ex vivo, and in vivo. Long-term effects on choroidal involution included a hypoxic outer neuroretina, associated with a progressive loss of RPE and photoreceptors, and visual deterioration. Early inhibition of IL-1ß receptor preserved choroid, decreased subretinal hypoxia, and prevented RPE/photoreceptor death, resulting in life-long improved visual function in IL-1 receptor antagonist-treated OIR animals. Together, these findings suggest a critical role for IL-1ß-induced choroidal degeneration in outer retinal dysfunction. Neonatal therapy using IL-1 receptor antagonist preserves choroid and prevents protracted outer neuroretinal anomalies in OIR, suggesting IL-1ß as a potential therapeutic target in ROP.

Mesenchymal Stromal Cells Promote Retinal Vascular Repair by Modulating Sema3E and IL-17A in a Model of Ischemic Retinopathy.

Ischemic retinopathies (IRs), such as retinopathy of prematurity and diabetic retinopathy, are characterized by an initial phase of microvascular degeneration that results in retinal ischemia, followed by exaggerated pathologic neovascularization (NV). Mesenchymal stromal cells (MSCs) have potent pro-angiogenic and anti-inflammatory properties associated with tissue repair and regeneration, and in this regard exert protection to neurons in ischemic and degenerative conditions; however, the exact mechanisms underlying these functions remain largely unknown. Class III Semaphorins (A-G) are particularly implicated in regulating neural blood supply (as well as neurogenesis) by suppressing angiogenesis and affecting myeloid cell function; this is the case for distinct neuropillin-activating Sema3A as well as PlexinD1-activating Sema3E; but during IR the former Sema3A increases while Sema3E decreases. We investigated whether retinal vascular repair actions of MSCs are exerted by normalizing Semaphorin and downstream cytokines in IR. Intravitreal administration of MSCs or their secretome (MSCs-conditioned media [MSCs-CM]) significantly curtailed vasoobliteration as well as aberrant preretinal NV in a model of oxygen-induced retinopathy (OIR). The vascular repair effects of MSCs-CM in the ischemic retina were associated with restored levels of Sema3E. Vascular benefits of MSCs-CM were reversed by anti-Sema3E; while intravitreal injection of anti-angiogenic recombinant Sema3E (rSema3E) in OIR-subjected mice reproduced effects of MSCs-CM by inhibiting as expected preretinal NV but also by decreasing vasoobliteration. To explain these opposing vascular effects of Sema3E we found in OIR high retinal levels, respectively, of the pro- and anti-angiogenic IL-17A and Sema3A-regulating IL-1ß; IL-17A positively affected expression of IL-1ß. rSema3E decreased concentrations of these myeloid cell-derived pro-inflammatory cytokines in vitro and in vivo. Importantly, IL-17A suppression by MSCs-CM was abrogated by anti-Sema3E neutralizing antibody. Collectively, our findings provide novel evidence by which MSCs inhibit aberrant NV and diminish vasoobliteration (promoting revascularization) in retinopathy by restoring (at least in part) neuronal Sema3E levels that reduce pathological levels of IL-17A (and in turn other proinflammatory factors) in myeloid cells. The ability of MSCs to generate a microenvironment permissive for vascular regeneration by controlling the production of neuronal factors involved in immunomodulatory activities is a promising opportunity for stem cell therapy in ocular degenerative 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.

Interleukin-1 Receptor Modulation Using ß-Substituted α-Amino-γ-Lactam Peptides From Solid-Phase Synthesis and Diversification.

As a key cytokine mediator of inflammation, interleukin-1ß (IL-1ß) binds to the IL-1 receptor (IL-1R) and activates various downstream signaling mediators, including NF-κB, which is required for immune vigilance and cellular protection. Toward the development of IL-1-targeting therapeutics which exhibit functional selectivity, the all-D-amino acid peptide 1 (101.10, H-D-Arg-D-Tyr-D-Thr-D-Val-D-Glu-D-Leu-D-Ala-NH2) was conceived as an allosteric IL-1R modulator that conserves NF-κB signaling while inhibiting other IL-1-activated pathways. Employing ß-hydroxy-α-amino-γ-lactam (Hgl) stereoisomers to study the conformation about the Thr3 residue in 1, [(3R,4S)-Hgl3]-1 (2b), among all possible diastereomers, was found to exhibit identical in vitro and in vivo activity as the parent peptide and superior activity to the α-amino-γ-lactam (Agl) counterpart. Noting the relevance of the ß-hydroxyl substituent and configuration for the activity of (3R,4S)-2b, fifteen different ß-substituted-Agl3 analogs of 1 (e.g., 2c-q) have now been synthesized by a combination of solution- and solid-phase methods employing N-Fmoc-ß-substituted-Agl3-Val-OH dipeptide building blocks. Introduction of a ß-azido-Agl3 residue into the resin bound peptide and subsequent reduction and CuAAC chemistry gave access to a series of amine and triazole derivatives (e.g., 2h-q). ß-Substituted-[Agl3]-1 analogs 2c-q exhibited generally similar circular dichroism (CD) spectra as that of Hgl analog 2b in water, presenting curve shapes indicative of ß-turn structures. The relevance of the ß-substituent was indicated in rodent models of preterm labor and retinopathy of prematurity (ROP), in which certain analogs inhibited preterm birth and vaso-obliteration, respectively, with activity similar to 1 and 2b. The ß-substituted-[Agl3]-1 analogs exhibited functional selectivity on IL-1-induced signaling pathways. The described solid-phase method has provided discerning probes for exploring peptide structure-activity relationships and valuable leads for developing prototypes to treat inflammatory events leading to prematurity and retinopathy of prematurity, which are leading causes of infant morbidity and blindness respectively.

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.

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.

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.

Erratum: Nuclear localization of platelet-activating factor receptor controls retinal neovascularization.

[This corrects the article DOI: 10.1038/celldisc.2016.17.].

Nuclear localization of platelet-activating factor receptor controls retinal neovascularization.

Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs.

Altered vascular function in fetal programming of hypertension.

BACKGROUND AND PURPOSE: Reduced endothelium-dependent vasorelaxation partly due to loss of nitric oxide (NO) bioavailability occurs in most cases of chronic hypertension. Intrauterine nutritional deprivation has been associated with increased risk for hypertension and stroke, associated with relaxant dysfunction and decreased vascular compliance, but the underlying mechanisms are not known. The present studies were undertaken to investigate whether endothelial dysfunction associated with altered NO-dependent vasodilatation pathways is also observed in a model of in utero programming of hypertension. METHODS: Pregnant Wistar rats were fed a normal (18%), low (9%), or very low (6%) protein isocaloric diet during gestation. Vasomotor response of resistance cerebral microvessels (<50 micro m) was studied in adult offspring of dams fed the 18% and 9% protein diets by a video imaging technique. Endothelial NOS (eNOS), soluble guanylate cyclase (sGC), and K(Ca) channel expression were measured by Western blot. NO synthase (NOS) activity was measured enzymatically as well as in situ by NADPH diaphorase staining. RESULTS: Litter size and survival to adulthood were not affected by the diets. Birth weights of offspring of dams fed the 6% diet were markedly lower than those of dams fed the 9% diet, which were marginally lower than those of controls. Systolic blood pressures of adult offspring of mothers in the 6% and 9% groups were comparably greater (156+/-2 and 155+/-1 mm Hg, respectively) than that of control offspring (137+/-1 mm Hg); we therefore focused on the 9% and 18% groups. Cerebral microvessel constriction to thromboxane A(2) mimetic and dilation to carba-prostaglandin I(2) did not differ between diet groups. In contrast, vasorelaxation to the NO-dependent agents substance P and acetylcholine was diminished by 50% in low protein-exposed offspring, but eNOS expression and activity were similar between the 2 diet groups. Vasorelaxant response to the NO donor sodium nitroprusside was also decreased and was associated with reduced (by 50% to 65%) cGMP levels and sGC expression. cGMP analogues caused comparable vasorelaxation in the 2 groups. Expression of K(Ca) (another important mediator of NO action) and relaxation to the K(Ca) opener NS1619 were unchanged by antenatal diet. CONCLUSIONS: Maternal protein deprivation, which leads to hypertension in the offspring, is associated with diminished NO-dependent relaxation of major organ (cerebral) microvasculature, which seems to be largely attributed to decreased sGC expression and cGMP levels. The study provides an additional explanation for abnormal vasorelaxation in nutrient-deprived subjects in utero.

Interleukin-1 and ischemic brain injury in the newborn: development of a small molecule inhibitor of IL-1 receptor.

Inflammation participates in the genesis and progression of hypoxic-ischemic brain injury. Interleukin (IL)-1 is a major pro-inflammatory cytokine, which plays a dominant role in hypoxic-ischemic (and postinfectious) brain damage. Abundant evidence reveals the principal involvement of IL-1 over other pro-inflammatory cytokines. IL-1 interacts with the IL-1 receptor I (IL-1RI). The natural IL-1 receptor antagonist (IL-1ra) is a large 17.5-kDa peptide that competes with IL-1 for its binding site on IL-1RI. Recombinant IL-1ra (Kineret) is effective in human inflammatory conditions. However, a number of drawbacks of IL-1ra limit its broader use; these include injection site reactions [70%], broad immunosuppression, and high costs. We hereby report the characterization of a small (peptide) IL-1RI antagonist we developed, namely rytvela (termed 101.10), and its efficacy in models of (gut) inflammation and of newborn hypoxic-ischemic brain injury. Experiments reveal that 101.10 is selective for the IL-1RI and inhibits to a variable extent different effects induced by IL-1. 101.10 is effective in vivo (on systemic as well as oral administration) in established models of inflammation involving IL-1, notably in inflammatory bowel disease, and is superior to dexamethasone. In a rat pup model of hypoxic-ischemic brain injury (Rice-Vannucci model), where IL-1 and IL-1R expression is increased, 101.10 preserved microvascular density, parenchymal integrity, and brain mass. In conclusion, we hereby describe for the first time the discovery of a stable, potent, and effective specific IL-1RI small (peptide) antagonist, namely 101.10 (rytvela), which exhibits allosteric modulatory properties, and is effective in vivo in models of inflammation (known to involve IL-1) and in particular in hypoxic-ischemic newborn brain injury. 101.10 (and small alike compounds) may be suitable alternatives to IL-1ra.

Development of a novel noncompetitive antagonist of IL-1 receptor.

IL-1 is a major proinflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein subunits. Currently available strategies to counter pathological IL-1 signaling rely on a recombinant IL-1 receptor antagonist, which directly competes with IL-1 for its binding site. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1R accessory protein, which are putative interactive sites with the IL-1RI subunit. In this study, we substantiate the merits of one of these peptides, rytvela (we termed "101.10"), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC(50) approximately 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed noncompetitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. Findings describe the discovery of a potent and specific small (peptide) antagonist of IL-1RI, with properties in line with an allosteric negative modulator.

Antenatal antioxidant prevents adult hypertension, vascular dysfunction, and microvascular rarefaction associated with in utero exposure to a low-protein diet.

Developmental programming of hypertension is associated with vascular dysfunction characterized by impaired vasodilatation to nitric oxide, exaggerated vasoconstriction to ANG II, and microvascular rarefaction appearing in the neonatal period. Hypertensive adults have indices of increased oxidative stress, and newborns that were nutrient depleted during fetal life have decreased antioxidant defenses and increased susceptibility to oxidant injury. To test the hypothesis that oxidative stress participates in early life programming of hypertension, vascular dysfunction, and microvascular rarefaction associated with maternal protein deprivation, pregnant rats were fed a normal, low protein (LP), or LP plus lazaroid (lipid peroxidation inhibitor) isocaloric diet from the day of conception until delivery. Lazaroid administered along with the LP diet prevented blood pressure elevation, enhanced vasomotor response to ANG II, impaired vasodilatation to sodium nitroprusside, and microvascular rarefaction in adult offspring. Liver total glutathione was significantly decreased in LP fetuses, and kidney eight-isoprostaglandin F2alpha (8-isoPGF(2alpha)) levels were significantly increased in adult LP offspring; these modifications were prevented by lazaroid. Renal nitrotyrosine abundance and blood levels of 1,4-dihydroxynonene and 4-hydroxynonenal-protein adducts were not modified by antenatal diet exposure. This study shows in adult offspring of LP-fed dams prevention of hypertension, vascular dysfunction, microvascular rarefaction, and of an increase in indices of oxidative stress by the administration of lazaroid during gestation. Lazaroid also prevented the decrease in antioxidant glutathione levels in fetuses, suggesting an antenatal mild oxidative stress in offspring of LP-fed dams. These studies support the concept that perinatal oxidative insult can lead to permanent alterations in the cardiovascular system development.

Dominant role for calpain in thromboxane-induced neuromicrovascular endothelial cytotoxicity.

Thromboxane A(2) (TXA(2)) is an important lipid mediator generated during oxidative stress and implicated in ischemic neural injury. This autacoid was recently shown to partake in this injury process by directly inducing endothelial cytotoxicity. We explored the mechanisms for this TXA(2)-evoked neural microvascular endothelial cell death. Stable TXA(2) mimetics 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-[1R-[1alpha,4alpha,5beta(Z),6alpha,(1E,3S)]]-9,11-dedioxy-9alpha,11alpha-methanolpoxy (U-46619) [as well as [1S-[1alpha,2alpha(Z),3beta(1E,3S(*)),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.1.1]-hept-2-yl]-5-heptenoic acid; I-BOP] induced a retinal microvascular degeneration in rat pups in vivo and in porcine retinal explants ex vivo and death of porcine brain endothelial cells (in culture). TXA(2) dependence of these effects was corroborated by antagonism using the selective TXA(2) receptor blocker (-)-6,8-difluoro-9-p-methyl-sulfonyl-benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid (L670596). In all cases, neurovascular endothelial cell death was prevented by pan-calpain and specific m-calpain inhibitors but not by caspase-3 or pan-caspase inhibitors. Correspondingly, TXA(2) (mimetics) augmented generation of known active m-calpain (but not mu-calpain) form and increased the activity of m-calpain (cleavage of fluorogenic substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; and of alpha-spectrin into specific fragments) but not of pan-caspase or specific caspase-3 (respectively, using sulforhodamine-Val-Arg-Asp-fluoromethyl ketone and detecting its active 17- and 12-kDa fragments). Interestingly, these effects were phospholipase C (PLC)-dependent [associated with increase in inositol triphosphate and inhibited by PLC blocker 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] and required calcium but were not associated with increased intracellular calcium. U-46619-induced calpain activation resulted in translocation of Bax to the mitochondria, loss of polarization of the latter (using potentiometric probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide; JC-1) and in turn release of cytochrome c into the cytosol and depletion of cellular ATP; these effects were all blocked by calpain inhibitors. Overall, this work identifies (specifically) m-calpain as a dominant protease in TXA(2)-induced neurovascular endothelial cell death.

Role of brain and peripheral angiotensin II in hypertension and altered arterial baroreflex programmed during fetal life in rat.

Intrauterine programming of hypertension is associated with evidence of increased renin-angiotensin system (RAS) activity. The current study was undertaken to investigate whether arterial baroreflex and blood pressure variability are altered in a model of in utero programming of hypertension secondary to isocaloric protein deprivation and whether activation of the RAS plays a role in this alteration. Pregnant Wistar rats were fed a normal-protein (18%) or low-protein (9%) diet during gestation, which had no effect on litter size, birth weight, or pup survival. Mean arterial blood pressure (MABP; 126 +/- 3 mm Hg 9% versus 108 +/- 4 mm Hg 18%; p < 0.05) and blood pressure variability were significantly greater in the adult offspring of the 9% protein-fed mothers. Arterial baroreflex control of heart rate, generated by graded i.v. infusion of phenylephrine and nitroprusside, was significantly shifted toward higher pressure; i.v. angiotensin-converting enzyme inhibitor normalized MABP and shifted the arterial baroreflex curve of the 9% offspring toward lower pressure without affecting the 18% offspring. For examining whether brain RAS is also involved in programming of hypertension, angiotensin-converting enzyme inhibitor and losartan (specific AT(1) receptor antagonist) were administered intracerebroventricularly; both significantly reduced MABP of the 9% but not the 18% offspring. Autoradiographic receptor binding studies demonstrated an increase in brain AT(1) expression in the subfornical organ and the vascular organ of the lamina terminalis in the 9% offspring. These data demonstrate a major tonic role of brain and peripheral RAS on hypertension associated with antenatal nutrient deprivation.