Chang He

Vasoprotective effect of PDGF-CC mediated by HMOX1 rescues retinal degeneration

Significance PDGF-CC plays critical roles in many biological processes, such as development, tumor growth, and angiogenesis. However, its role in blood vessel survival/regression and the underlying mechanisms remain unknown. Here, using different loss- and gain-of-function assays and multiple model systems, we show that PDGF-CC is a critical vascular protective factor required to maintain blood vessel survival. Mechanistically, we found that heme oxygenase-1 (HMOX1) activity is crucial for the vascular protective/survival effect of PDGF-CC. Given the general involvement of vascular degeneration in most degenerative diseases, PDGF-CC may be of therapeutic use in treating different types of degenerative disorders. Our findings point out that the PDGF-CC level should be monitored closely in various pathological conditions to ensure normal blood vessel survival. Blood vessel degeneration is critically involved in nearly all types of degenerative diseases. Therefore strategies to enhance blood vessel protection and survival are highly needed. In this study, using different animal models and cultured cells, we show that PDGF-CC is a potent vascular protective and survival factor. PDGF-CC deficiency by genetic deletion exacerbated blood vessel regression/degeneration in various animal models. Importantly, treatment with PDGF-CC protein not only increased the survival of retinal blood vessels in a model of oxygen-induced blood vessel regression but also markedly rescued retinal and blood vessel degeneration in a disease model of retinitis pigmentosa. Mechanistically, we revealed that heme oxygenase-1 (HMOX1) activity is critically required for the vascular protective/survival effect of PDGF-CC, because blockade of HMOX1 completely abolished the protective effect of PDGF-CC in vitro and in vivo. We further found that both PDGF receptors, PDGFR-β and PDGFR-α, are required for the vasoprotective effect of PDGF-CC. Thus our data show that PDGF-CC plays a pivotal role in maintaining blood vessel survival and may be of therapeutic value in treating various types of degenerative diseases.

Angiogenesis Mediated by Toll-Like Receptor 4 in Ischemic Neural Tissue

Objective—Activation of the immune system via toll-like receptors (TLRs) is implicated in atherosclerosis, microvascular complications, and angiogenesis. However, the involvement of TLRs in inflammation-associated angiogenesis in ischemic neural tissue has not been investigated. The goal of this study is to determine the role of TLR4 signaling in oxygen-induced neovascularization in retina, a neural tissue. Methods and Results—In oxygen-induced retinopathy model, we found that retinal neovascularization was significantly attenuated in TLR4−/− mice. The further study revealed that the absence of TLR4 led to downregulation of proinflammatory factors in association with the attenuated activation of glia in the ischemic retina, which was also associated with reduced expression of high-mobility group box-1, an endogenous ligand for TLR4. The application of high-mobility group box-1 to the ischemic retina promoted the production of proinflammatory factors in wild-type but not TLR4−/− mice. High-mobility group box-1 treatment in vitro also significantly promoted the production of proinflammatory factors in retinal glial cells from wild-type mice, but much less from TLR4−/− mice. Conclusion—Our results suggest that the release of high-mobility group box-1 in ischemic neural tissue initiates TLR4-dependent responses that contribute to neovascularization. These findings represented a previously unrecognized effect of TLR4 on angiogenesis in association with the activation of glia in ischemic neural tissue.

Objective evaluation of the changes in the crystalline lens during accommodation in young and presbyopic populations using Pentacam HR system.

AIM To quantify the changes in the lens profile with accommodation in different age groups. METHODS The Pentacam HR system was used to obtain the images of the anterior eye segment from 23 young and 15 presbyopic emmetropic subjects in unaccommodated (with an accommodation stimulus of 0.0D) and accommodated (with an accommodation stimulus of 5.0D for the young group and 1.0D for the presbyopic group) states. The phakic crystalline lens shape, including curvature of crystalline lens and central lens thickness (CLT), and the measurements of anterior segment length (ASL), central anterior chamber depth (CACD) were investigated. The anterior chamber volume (ACV) was also measured. RESULTS The reduction of CACD and ACV were significant in both groups after accommodation stimulus. From the profile of anterior eye segment, a significant decrease in anterior crystalline lens radii of curvature (-2.52mm) and a mean increase in CLT (0.222mm) and ASL (0.108mm) were found in the young group with an accommodation stimulus of 5.0D. However, no statistically significant changes of CLT, ASL, or crystalline lens radii of curvature were found in the presbyopic group. CONCLUSION Our data showed that the shallowing of anterior chamber during accommodation was caused by the forward bulging of the anterior lens surface, rather than by anterior shifting of lens position in either young or presbyopic subjects.

Necroptosis in microglia contributes to neuroinflammation and retinal degeneration through TLR4 activation

Inflammation has emerged to be a critical mechanism responsible for neural damage and neurodegenerative diseases. Microglia, the resident innate immune cells in retina, are implicated as principal components of the immunological insult to retinal neural cells. The involvement of microglia in retinal inflammation is complex and here we propose for the first time that necroptosis in microglia triggers neuroinflammation and exacerbates retinal neural damage and degeneration. We found microglia experienced receptor-interacting protein kinase 1 (RIP1)- and RIP3-dependent necroptosis not only in the retinal degenerative rd1 mice, but also in the acute retinal neural injury mice. The necroptotic microglia released various pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-α and chemokine (C-C motif) ligand 2, which orchestrated the retinal inflammation. Importantly, necroptosis blockade using necrostatin-1 could suppress microglia-mediated inflammation, rescue retinal degeneration or prevent neural injury in vivo. Meanwhile, cultured microglia underwent RIP1/3-mediated necroptosis and the necroptotic microglia produced large amounts of pro-inflammatory cytokines in response to lipopolysaccharide or oxidative stress in vitro. Mechanically, TLR4 deficiency ameliorated microglia necroptosis with decreased expression levels of machinery molecules RIP1 and RIP3, and suppressed retinal inflammation, suggesting that TLR4 signaling was required in microglia necroptosis-mediated inflammation. Thus, we proposed that microglia experienced necroptosis through TLR4 activation, promoting an inflammatory response that serves to exacerbate considerable neural damage and degeneration. Necroptosis blockade therefore emerged as a novel therapeutic strategy for tempering microglia-mediated neuroinflammation and ameliorating neural injury and neurodegenerative diseases.Inflammation has emerged to be a critical mechanism responsible for neural damage and neurodegenerative diseases. Microglia, the resident innate immune cells in retina, are implicated as principal components of the immunological insult to retinal neural cells. The involvement of microglia in retinal inflammation is complex and here we propose for the first time that necroptosis in microglia triggers neuroinflammation and exacerbates retinal neural damage and degeneration. We found microglia experienced receptor-interacting protein kinase 1 (RIP1)- and RIP3-dependent necroptosis not only in the retinal degenerative rd1 mice, but also in the acute retinal neural injury mice. The necroptotic microglia released various pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-α and chemokine (C-C motif) ligand 2, which orchestrated the retinal inflammation. Importantly, necroptosis blockade using necrostatin-1 could suppress microglia-mediated inflammation, rescue retinal degeneration or prevent neural injury in vivo. Meanwhile, cultured microglia underwent RIP1/3-mediated necroptosis and the necroptotic microglia produced large amounts of pro-inflammatory cytokines in response to lipopolysaccharide or oxidative stress in vitro. Mechanically, TLR4 deficiency ameliorated microglia necroptosis with decreased expression levels of machinery molecules RIP1 and RIP3, and suppressed retinal inflammation, suggesting that TLR4 signaling was required in microglia necroptosis-mediated inflammation. Thus, we proposed that microglia experienced necroptosis through TLR4 activation, promoting an inflammatory response that serves to exacerbate considerable neural damage and degeneration. Necroptosis blockade therefore emerged as a novel therapeutic strategy for tempering microglia-mediated neuroinflammation and ameliorating neural injury and neurodegenerative diseases.

Microglia Polarization with M1/M2 Phenotype Changes in rd1 Mouse Model of Retinal Degeneration

Microglia activation is recognized as the hallmark of neuroinflammation. However, the activation profile and phenotype changes of microglia during the process of retinal degeneration are poorly understood. This study aimed to elucidate the time-spatial pattern of microglia distribution and characterize the polarized phenotype of activated microglia during retinal neuroinflammation and degeneration in rd1 (Pde6βrd1/rd1) mice, the classic model of inherited retinal degeneration. Retinae of rd1 mice at different postnatal days (P7, P14, P21, P28, P56, and P180) were prepared for further analysis. We found most CD11b+ or IBA1+ microglia expressed Ki-67 and CD68 in rd1 mice and these cells migrated toward the layer of degenerative photoreceptors at the rapid rods degeneration phase from P14 to P28. These microglia exhibited typical ameboid activated shape with round bodies and scarce dendrites, while at late phase at P180, they displayed resting ramified morphology with elongated dendrites. Flow cytometry revealed that the percentage of CD86+CD206- M1 microglia increased markedly in rd1 retinae, however, no significant change was observed in CD206+CD86- M2 microglia. Interestingly, CD86+CD206+ microglia, an intermediate state between the two extremes of M1 and M2, increased markedly at the rapid rods degeneration phase. The immunofluorescence images revealed that microglia in rd1 mice highly expressed M1 markers including CD16/32, CD86, and CD40. In addition, increased expression of pro-inflammatory cytokines (TNF-α, IL-6, and CCL2) was observed in rd1 mice. Our findings unfolded a panorama for the first time that microglia conducted distinctive behaviors with the progression of retinal degeneration in rd1 mice. Microglia is activated and particularly polarized to a pro-inflammatory M1 phenotype at the rapid rods degenerative phase, suggesting that the involvement of M1 microglia in the retinal neuroinflammation and degeneration. Most microglia adopted an intermediate polarization “M1½” state in rd1, revealing that microglia orchestrated a complicated continuous spectrum in degenerative retina.

Autophagy regulates TGF-β2-induced epithelial-mesenchymal transition in human retinal pigment epithelium cells

Transforming growth factor (TGF)-β2-induced epithelial-mesenchymal transition (EMT) in human retinal pigment epithelium (RPE) cells has an important role in the pathophysiology of intraocular fibrotic disorders, which may cause vision impairment and blindness. Autophagy, an intracellular homeostatic pathway, contributes to the physiological and pathological processes of RPE. Furthermore, autophagy has previously been reported to function in the EMT process in numerous tissue and cell types. However, the association between autophagy and the EMT process in RPE cells has not yet been fully determined. The present study demonstrated that TGF-β2-treated human RPE cells (ARPE-19 cell line) exhibited a significantly increased autophagic flux compared with control cells, as determined by western blot analysis of the protein levels of microtubule-associated protein 1 light chain 3-II and p62 (also termed sequestosome 1). Furthermore, it was demonstrated that autophagy activation enhanced the TGF-β2-induced EMT process in ARPE-19 cells, and inhibition of autophagy by chloroquine administration attenuated TGF-β2-induced EMT, which was determined by analyzing the expression of mesenchymal and epithelial markers by reverse transcription-quantitative polymerase chain reaction and/or western blotting. A transwell migration and invasion assays was also performed that demonstrated that autophagy activation by rapamycin enhanced TGF-β2-stimulated RPE cell migration and invasion, and inhibition of autophagy reduced TGF-β2-stimulated RPE cell migration and invasion. These results also demonstrated that autophagy activation enhanced the TGF-β2-induced EMT process in ARPE-19 cells, and inhibition of autophagy attenuated TGF-β2-induced EMT. Overall, the results of the present study demonstrated that TGF-β2-induced EMT may be regulated by autophagy, thus indicating that autophagy may serve as a potential therapeutic target for the attenuation of EMT in intraocular fibrotic disorders.

Alpha-1 Antitrypsin Attenuates M1 Microglia-Mediated Neuroinflammation in Retinal Degeneration

Neurodegenerative diseases are a set of disorders characterized by progressive neuronal death and are associated with microglia-mediated neuroinflammation. Recently, neuroinflammation is proposed as a promising therapeutic target for many neurodegenerative diseases. Alpha-1 antitrypsin (AAT) is recognized as a novel immunomodulatory agent in autoimmune diseases and transplantation, however, its impact on neuroinflammation and neurodegeneration remains unknown. This study aims to explore the effects of AAT on microglia-mediated neuroinflammation and retinal degeneration in rd1 mouse model. We found reduced expression of AAT in rd1 retina, and AAT supplement exhibited certain protective effect on retinal degeneration, presenting with increased amount of photoreceptor nuclei, and amplified wave amplitudes in electroretinogram analysis. Of note, AAT shifted microglia phenotype from pro-inflammatory M1 (CD16/CD32+, iNOS+) to anti-inflammatory M2 (CD206+, Arg1+) both in vivo and in vitro, underscoring the concept of immunomodulation on microglia polarization by AAT during neurodegeneration. Furthermore, AAT suppressed the activation of STAT1, promoted the expression of IRF4 while inhibited IRF8 expression, indicating the involvement of these signaling pathways in AAT immunomodulation. Collectively, our data provided evidence for a novel protective role of AAT through immunomodulation on microglia polarization. Attenuating neuroinflammation by AAT may be beneficial to retard neurodegeneration in rd1 mice.

Imatinib Ameliorated Retinal Neovascularization by Suppressing PDGFR-α and PDGFR-β

Background/Aims: Platelet-derived growth factors (PDGFs) have emerged as pivotal in pathological angiogenesis, which is a hallmark of various tumors and retinal diseases. Here we evaluated the anti-angiogenic effect of imatinib, an inhibitor of PDGF receptors α and β (PDGFR-α and -β), in retinal neovascularization using an oxygen-induced retinopathy (OIR) model. Methods: The OIR model was established and given imatinib or vehicle treatments daily from P12 to P16. At the peak of angiogenesis at P17, the neovascularization area was quantified on retinal whole-mounts with isolectin B4 staining. Immunofluorescence staining and western blots were used to determine the effect of imatinib on different vascular cells and the pathway molecules involved. Results: Imatinib effectively suppressed pathological angiogenesis in OIR mice and reduced the number of all three types of vascular cells, including endothelial cells, pericytes, and smooth muscle cells. Moreover, the expression and activation of PDGFR-α and -β were inhibited by imatinib. The imatinib-treated OIR mice presented with reduced expression of other potent pro-angiogenic factors such as VEGF and FGF2. No obvious retinal or systemic side effects were observed in the imatinib treatment group. Conclusions: Imatinib appears to be safe and effective in suppressing retinal neovascularization. Targeting PDGFs/PDGFRs may also be important for anti-angiogenic treatment and offer a viable alternative treatment for retinal angiogenic diseases.