Inhibition of Reactive Gliosis Prevents Neovascular Growth in the Mouse Model of Oxygen-Induced Retinopathy

Retinal neovascularization (NV) is a major cause of blindness in ischemic retinopathies. Previous investigations have indicated that ischemia upregulates GFAP and PDGF-B expression. GFAP overexpression is a hallmark of reactive gliosis (RG), which is the major pathophysiological feature of retinal damage. In addition, PDGF-B has been implicated in proliferative retinopathies. It was the aim of this study to gain insights on the possible pharmacological interventions to modulate PDGF-B and GFAP expression, and its influence on RG and NV. We used an array of assays to evaluate the effects of YC-1, a small molecule inhibitor of HIF-1 and a novel NO-independent activator of soluble guanylyl cyclase (sGC), on RG and NV, in vivo and in vitro. When compared to the DMSO-treated retinas, dual-intravitreal injections of YC-1, in vivo: (1) suppressed the development and elongation of neovascular sprouts in the retinas of the oxygen-induced retinopathy (OIR) mouse model; and (2) reduced ischemia-induced overexpression of GFAP and PDGF-B at the message (by 64.14±0.5% and 70.27±0.04%) and the protein levels (by 65.52±0.02% and 57.59±0.01%), respectively. In addition, at 100 µM, YC-1 treatment downregulated the hypoxia-induced overexpression of GFAP and PDGF-B at the message level in rMC-1 cells (by 71.42±0.02% and 75±0.03%), and R28 cells (by 58.62±0.02% and 50.00±0.02%), respectively; whereas, the protein levels of GFAP and PDGF-B were reduced (by 78.57±0.02% and 77.55±0.01%) in rMC-1cells, and (by 81.44±0.02% and 79.16±0.01%) in R28 cells, respectively. We demonstrate that YC-1 reversed RG during ischemic retinopathy via impairing the expression of GFAP and PDGF-B in glial cells. This is the first investigation that delves into the reversal of RG during ischemic retinal vasculopathies. In addition, the study reveals that YC-1 may exert promising therapeutic effects in the treatment of retinal and neuronal pathologies

Deletion of low molecular weight protein tyrosine phosphatase (Acp1) protects against stress-induced cardiomyopathy.

The low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene, is a ubiquitously expressed phosphatase whose in vivo function in the heart and in cardiac diseases remains unknown. To investigate the in vivo role of LMPTP in cardiac function, we generated mice with genetic inactivation of the Acp1 locus and studied their response to long-term pressure overload. Acp1(-/-) mice develop normally and ageing mice do not show pathology in major tissues under basal conditions. However, Acp1(-/-) mice are strikingly resistant to pressure overload hypertrophy and heart failure. Lmptp expression is high in the embryonic mouse heart, decreased in the postnatal stage, and increased in the adult mouse failing heart. We also show that LMPTP expression increases in end-stage heart failure in humans. Consistent with their protected phenotype, Acp1(-/-) mice subjected to pressure overload hypertrophy have attenuated fibrosis and decreased expression of fibrotic genes. Transcriptional profiling and analysis of molecular signalling show that the resistance of Acp1(-/-) mice to pathological cardiac stress correlates with marginal re-expression of fetal cardiac genes, increased insulin receptor beta phosphorylation, as well as PKA and ephrin receptor expression, and inactivation of the CaMKIIδ pathway. Our data show that ablation of Lmptp inhibits pathological cardiac remodelling and suggest that inhibition of LMPTP may be of therapeutic relevance for the treatment of human heart failure

The nexus between VEGF and NFκB orchestrates a hypoxia-independent neovasculogenesis.

Nuclear Factor-Kappa B [NFκB] activation triggers the elevation of various pro-angiogenic factors that contribute to the development and progression of diabetic vasculopathies. It has been demonstrated that vascular endothelial growth factor [VEGF] activates NFκB signaling pathway. Under the ischemic microenvironments, hypoxia-inducible factor-1 [HIF-1] upregulates the expression of several proangiogenic mediators, which play crucial roles in ocular pathologies. Whereas YC-1, a soluble guanylyl cyclase [sGC] agonist, inhibits HIF-1 and NFκB signaling pathways in various cell and animal models. Throughout this investigation, we examined the molecular link between VEGF and NFκB under a hypoxia-independent microenvironment in human retinal microvascular endothelial cells [hRMVECs]. Our data indicate that VEGF promoted retinal neovasculogenesis via NFκB activation, enhancement of its DNA-binding activity, and upregulating NFκB/p65, SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9 expression. Conversely, YC-1 impaired the activation of NFκB and its downstream signaling pathways, via attenuating IκB kinase phosphorylation, degradation and activation, and thus suppressing p65 phosphorylation, nuclear translocation, and inhibiting NFκB-DNA binding activity. We report for the first time that the nexus between VEGF and NFκB is implicated in coordinating a scheme that upregulates several pro-angiogenic molecules, which promotes retinal neovasculogenesis. Our data may suggest the potential use of YC-1 to attenuate the deleterious effects that are associated with hypoxia/ischemia-independent retinal vasculopathies

Immunohistochemical Analysis of PDGF-B, <i>in vivo</i>.

<p>Photomicrographs of retinas from various OIR groups that were immunostained for PDGF-B. The expression of PDGF-B was upregulated in the non-treated ischemic and DMSO-treated groups, compared with non-treated normoxic group. While all protein immunoreactivities were downregulated in the YC-1-treated group, compared with DMSO-treated groups. Data are representative of 3 independent experiments. Scale bar: 140 µm.</p

YC-1 Restrains Hypoxia-Induced Upregulation of GFAP and PDGF-B Protein Levels, in Glial Cells.

<p>Western Blot analysis indicated that protein expression levels were elevated markedly in the non-treated hypoxic cells [A]. In YC-1-treated hypoxic cells, GFAP and PDGF-B protein expression were significantly decreased in a dose-dependent fashion, compared with DMSO-treated hypoxic cells. Statistical significance was determined by ANOVA [**<i>P</i><0.01]. Data are representative of 3 independent experiments. The densitometric analysis of Western Blot data [B] represents the intensities of GFAP and PDGF-B protein expressions in rMC-1 and R28 cells relative to those of β-actin expression, whereas the relative ratio of hypoxia control was defined as 100. Values, shown as the mean ± SEM, from 3 separate experiments with a total sample size of 6. [**<i>P</i><0.01 as compared to DMSO-treated hypoxic control].</p

YC-1 Exhibits Anti-Reactive Gliosis and Anti-Angiogenic Properties, <i>in vivo</i> and <i>in vitro</i>.

<p>Assessments of retinal immunohistochemical analysis exhibit the values that were obtained from at least 5 retinal fields were used to calculate the average pixel intensity value/retina [A]. Bar graphs exhibit the area of staining of GFAP and PDGF-B in all four groups. Values [mean ± SEM], from 3 separate experiments from at least 10 images from 4 different eyes/group. [***<i>P</i><0.001 and **<i>P</i><0.01 as compared to DMSO-treated retinas]. [B] Coculture models. [<i>i</i>] Cell proliferation assay model. [<i>ii</i>] Cell migration assay model; were both utilized to investigate the effects of YC-1 on the proliferation [C] and migration [D] of ECs in a coculture system. hRMVECs growth curves from four groups were depicted. Coculture group had a higher proliferation and migration rate of hRMVECs cells than that of hRMVECs solo [<i>**P<0.01</i>]. Hypoxia significantly increased hRMVECs proliferation and migration rate in the coculture system [<i>**P<0.01</i>, rMC-1/hypoxia vs. rMC-1/normoxia]. After rMC-1 cells were treated with YC-1 [100 µM], the proliferation and the migration rate of hRMVECs were significantly inhibited compared with the rMC-1/hypoxia group. [<i>**P<0.01</i>, YC-1-treated rMC-1/hypoxia vs. nontreated rMC-1/hypoxia]. Data are representative of 3 independent experiments.</p

Analyses of Retinal Vascular Development in the Normal Retinal Vasculature and the Neovascular Retina.

<p>Mice were perfused with fluorescein-labeled dextran. Normal mice were analyzed P2 [A], P4 [B], and P7 [C]. Exposure of mice to 75% O₂ between P7–P12 led to rapidly progressive vaso-obliteration of the retinal vasculature over. Retinal NV was analyzed in the OIR mice on P12 [D], P15 (OIR) [E], and P17 (OIR) [F]. [n = 15 per group]. bar: 300 µm.</p

Immunofluorescence Analysis of GFAP Expression, <i>in vitro</i>.

<p>Photomicrographs show the rMC-1 and R28 cells, which were immunostained with anti-GFAP antibody. Intense staining was considered a positive signal. Yellow arrows indicate GFAP immunostaining. Under hypoxia, non-treated hypoxic cells exhibited extremely high GFAP immunoreactivity. Treatment of cells with YC-1 [25–100 µM] under hypoxia for 48 hours resulted in a dose-dependent inhibition of GFAP expression. Images are representatives of 3 independent experiments. Scale bar: 40 µm. Graphs in the bottom represent the assessments of the cellular immunocytochemical analysis of GFAP. Graphs showing the intensity of GFAP staining in rMC-1 and R28 cells after treatment with YC-1 relative to that measured in DMSO-treated hypoxic control. The areas of staining to GFAP/µm² in all four groups were measured. Values, shown as the mean ± SEM, from 3 separate experiments.</p

The Influence of YC-1 on Neovascular Sprouts Number and Length.

<p>Images exhibit a higher magnification of growing tips of BVs that reveal multiple neovascular sprouts growing at multiple angles including both along and above the plane of the section. Image along the leading edge of vascularization from; [A] Non-treated ischemic retinas; [B] DMSO-treated retinas; [C, D, E, and F] represent retinas from animals that were injected with YC-1 [25–100 µl]. neovascular sprouts lengths and numbers were not influenced by DMSO treatment. Whereas treatment with YC-1 significantly and dose-dependently inhibited the number and the length of neovascular sprouts. [n = 5 per group].</p

YC-1 Reverses Retinal Reactive Gliosis and Retinal Neovascularization, <i>in vivo</i> and <i>in vitro</i>.

<p>Neovascular sprouts from DMSO-treated retinas had approximately similar lengths as the non-treated ischemic retinas [A]. YC-1 treated animals exhibited a significant and a dose-dependent decrease in neovascular sprouts lengths as compared to DMSO-treated retinas. [ANOVA, *<i>**P<0.001</i>; <i>**P<0.01</i> between YC-1 and DMSO] [A]. DMSO has no influence on the number of neovascular sprouts [B]. Whereas YC-1 exhibited a significant and a dose-dependent decrease in neovascular sprouts number [B]. [ANOVA, *<i>**P<0.001</i>; <i>**P<0.01</i> between YC-1 and DMSO]. There were 24 neovascular sprouts/retina that were averaged. [n = 5 per group]. By using primer sets [C]; Real Time RT-PCR analyses, <i>in vivo</i> [D and E] indicate that the levels of <i>GFAP</i> and <i>PDGF-B</i> mRNA were increased in the non-treated ischemic retinas, while non-treated normoxic retinas exhibited extremely low levels. Treatment of ischemic retinas with dual injections of YC-1 resulted in a significant knockdown of PDGF-B [**<i>P</i><0.01] and GFAP [**<i>P</i><0.01] gene expression when compared with DMSO-treated controls. ANOVA; Mean ± SEM of mRNA level normalized to β-actin were calculated, [***<i>P</i><0.001 and **<i>P</i><0.01, as compared to DMSO-treated retinas]. Data are representative of 3 independent experiments. Real Time RT-PCR analysis, <i>in vitro</i> [F] indicate that post-culturing R28 and rMC-1 cells under normoxic and hypoxic conditions; the mRNA levels of <i>GFAP and PDGF-B</i> were upregulated in all non-treated hypoxic cells, while normoxic cells exhibited remarkable low mRNA levels. Treatment of hypoxic R28 and rMC-1 cells with various concentrations of YC-1 resulted in a significant inhibition of <i>GFAP and PDGF-B</i>, mRNA expression as compared to DMSO-treated controls. ANOVA was used for statistical analyses. Mean ± SEM of mRNA level normalized to β-actin were calculated, [***<i>P</i><0.001 and **<i>P</i><0.01 as compared to DMSO-treated hypoxic control. Data are representative of 3 independent experiments].</p