Accumulation of damaged or misfolded proteins resulted from oxidative protein modification induces endoplasmic reticulum (ER) stress by activating the pathways of unfolded protein response. In pathologic hemolytic conditions, extracellular free hemoglobin is submitted to rapid oxidation causing heme release. Resident cells of atherosclerotic lesions, after intraplaque hemorrhage, are exposed to heme leading to oxidative injury. Therefore, we raised the question whether heme can also provoke ER stress. Smooth muscle cells are one of the key players of atherogenesis; thus, human aortic smooth muscle cells (HAoSMCs) were selected as a model cell to reveal the possible link between heme and ER stress. Using immunoblotting, quantitative polymerase chain reaction and immunocytochemistry, we quantitated the markers of ER stress. These were: phosphorylated eIF2α, Activating transcription factor-4 (ATF4), DNA-damage-inducible transcript 3 (also known as C/EBP homology protein, termed CHOP), X-box binding protein-1 (XBP1), Activating transcription factor-6 (ATF6), GRP78 (glucose-regulated protein, 78kDa) and heme responsive genes heme oxygenase-1 and ferritin. In addition, immunohistochemistry was performed on human carotid artery specimens from patients who had undergone carotid endarterectomy. We demonstrate that heme increases the phosphorylation of eiF2α in HAoSMCs and the expression of ATF4. Heme also enhances the splicing of XBP1 and the proteolytic cleavage of ATF6. Consequently, there is up-regulation of target genes increasing both mRNA and protein levels of CHOP and GRP78. However, TGFβ and collagen type I decreased. When the heme binding proteins, alpha-1-microglobulin (A1M) and hemopexin (Hpx) are present in cell media, the ER stress provoked by heme is inhibited. ER stress pathways are also retarded by the antioxidant N-acetyl cysteine (NAC) indicating that reactive oxygen species are involved in heme-induced ER stress. Consistent with these findings, elevated expression of the ER stress marker GRP78 and CHOP were observed in smooth muscle cells of complicated lesions with hemorrhage compared to either atheromas or healthy arteries. In conclusion, heme triggers ER stress in a time- and dose-dependent manner in HAoSMCs. A1M and Hpx as well as NAC effectively hamper heme-induced ER stress, supporting their use as a potential therapeutic approach to reverse such a deleterious effects of heme toxicity.

Diabetic retinopathy (DR) is the leading cause of vision loss among working-age adults. The interplay between hyperglycemia and endothelial activation in inducing endoplasmic reticulum (ER) stress pathways and visual deficits in DR is not fully understood. To address this, we used a mouse model of chronic vascular activation using endothelial-specific tumor necrosis factor-α (TNF-α)-expressing (tie2-TNF) mice to induce diabetes with streptozotocin. At 4 weeks post streptozotocin, a significant 2-fold to 10-fold increase in retinal neurovascular inflammatory gene transcript response in tie2-TNF mice was further increased in diabetic tie2-TNF mice. A decrease in visual acuity and scotopic b-wave amplitude in tie2-TNF mice was further accentuated in diabetic tie2-TNF mice and these changes correlated with a multi-fold increase in retinal ER stress markers and a reduction in adherens junctions. Cultured retinal endothelial cells showed a significant decrease in trans-endothelial resistance as well as VE-cadherin expression under TNF-α and high glucose stress. These changes were partly rescued by tauroursodeoxycholic acid, a potent ER stress inhibitor. Taken together, constant endothelial activation induced by TNF-α further exacerbated by hyperglycemia results in activation of ER stress and chronic proinflammation in a feed forward loop ultimately resulting in endothelial junction protein alterations leading to visual deficits in the retina. Inhibition of ER stress and endothelial activation may prove to be a novel therapeutic target in DR.

Increased O-GlcNAcylation, a well-known post-translational modification of proteins causally linked to various detrimental cellular functions in pathological conditions including diabetic retinopathy (DR). Previously we have shown that endothelial activation induced by inflammation and hyperglycemia results in the endoplasmic reticulum (ER) stress-mediated intercellular junction alterations accompanied by visual deficits in a tie2-TNF-α transgenic mouse model. In this study, we tested the hypothesis that increased ER stress via O-GlcNAcylation of VE-Cadherin likely contribute to endothelial permeability. We show that ER stress leads to GRP78 translocation to the plasma membrane, increased O-GlcNAcylation of proteins, particularly VE-Cadherin resulting in a defective complex partnering leading to the loss of retinal endothelial barrier integrity and increased transendothelial migration of monocytes. We further show an association of GRP78 with the VE-Cadherin under these conditions. Interestingly, cells exposed to ER stress inhibitor, tauroursodeoxycholic acid partially mitigated all these effects. Our findings suggest an essential role for ER stress and O-GlcNAcylation in altering the endothelial barrier function and reveal a potential therapeutic target in the treatment of DR.

Objectives: To evaluate our experience with a significant number of brain malignancy–related vocal fold paralysis patients and their response to vocal cord–related therapies. Background: Vocal fold paralysis is a potentially devastating complication of various types of pediatric diseases and surgeries that can lead to significant vocal and swallowing difficulties. While there is significant data in the literature on outcomes of children treated for vocal fold paralysis following cardiac or thyroid surgery, there is a scarcity of such information on children following the treatment of neurologic malignancy. Methods: Records of 19 patients at a tertiary center who were treated for neurologic malignancies and developed either unilateral or bilateral vocal fold paralysis were reviewed for vocal fold pathology and vocal fold paralysis treatment-related variables, including initial diagnosis, management with observation or speech therapy, duration of therapy, pre- and postintervention swallow studies, and surgical intervention. Results: Bilateral vocal fold paralysis was noted in 26% (5/19) patients. Eighty-four percent (16/19) of patients had stable or improved ability to vocalize and swallow following therapy. There was no statistically significant difference in speech or swallowing improvement after speech therapy alone or speech therapy in combination with injection laryngoplasty ( P = .25). No complications were noted with surgical intervention. Conclusions: Patients with vocal fold paralysis secondary to neurologic malignancy can have an improvement in speech and swallowing after a variety of treatments, including speech therapy or early injection laryngoplasty. There was no statistically significant difference in improvement based on the type of intervention utilized. A larger sample size is needed to conclude whether surgical intervention combined with speech therapy leads to more rapid and significant functional improvement than speech therapy alone.

Stress-associated premature senescence plays a major role in retinal diseases. In this study, we investigated the relationship between endothelial dysfunction, endoplasmic reticulum (ER) stress, and cellular senescence in the development of retinal dysfunction. We tested the hypothesis that constant endothelial activation by transmembrane tumor necrosis factor-α (tmTNF-α) exacerbates age-induced visual deficits via senescence-mediated ER stress in this model. To address this, we employed a mouse model of chronic vascular activation using endothelial-specific TNF-α-expressing (tie2-TNF) mice at 5 and 10 months of age. Visual deficits were exhibited by tie2-TNF mice at both 5 months and 10 months of age, with the older mice showing statistically significant loss of visual acuity compared with tie2-TNF mice at age 5 months. The neural defects, as measured by electroretinogram (ERG), also followed a similar trend in an age-dependent fashion, with 10-month-old tie2-TNF mice showing the greatest decrease in "b" wave amplitude at 25 cd.s.m compared with age-matched wildtype (WT) mice and five-month-old tie2-TNF mice. While gene and protein expression from the whole retinal extracts demonstrated increased inflammatory (Icam1, Ccl2), stress-associated premature senescence (p16, p21, p53), and ER stress (Grp78, p-Ire1α, Chop) markers in five-month-old tie2-TNF mice compared with five-month-old WT mice, a further increase was seen in 10-month-old tie2-TNF mice. Our data demonstrate that tie2-TNF mice exhibit age-associated increases in visual deficits, and these data suggest that inflammatory endothelial activation is at least partly at play. Given the correlation of increased premature senescence and ER stress in an age-dependent fashion, with the loss of visual functions and increased endothelial activation, our data suggest a possible self-enhanced loop of unfolded protein response pathways and senescence in propagating neurovascular defects in this model. Impact statement Vision loss in most retinal diseases affects the quality of life of working age adults. Using a novel animal model that displays constant endothelial activation by tmTNF-α, our results demonstrate exacerbated age-induced visual deficits via premature senescence-mediated ER stress. We have compared mice of 5 and 10 months of age, with highly relevant human equivalencies of approximately 35- and 50-year-old patients, representing mature adult and middle-aged subjects, respectively. Our studies suggest a possible role for a self-enhanced loop of ER stress pathways and senescence in the propagation of retinal neurovascular defects, under conditions of constant endothelial activation induced by tmTNF-α signaling.