Tauroursodeoxycholic Acid Alleviates Endoplasmic Reticulum Stress-Mediated Visual Deficits in Diabetic tie2-TNF Transgenic Mice via TGR5 Signaling.

Purpose: This study evaluated if tauroursodeoxycholic acid (TUDCA) alleviates pro-inflammatory and endoplasmic reticulum (ER) stress-mediated visual deficits in diabetic tie2-TNF transgenic mice via Takeda G protein-coupled receptor 5 (TGR5) receptor signaling. Methods: Adult tie2-TNF transgenic or age-matched C57BL/6J (wildtype, WT) mice were made diabetic and treated subcutaneously with TUDCA. After 4 weeks, visual function, vascular permeability, immunohistology, and molecular analyses were assessed. Human retinal endothelial cells (HRECs) silenced for TGR5, followed by TNF and high glucose (HG) stress-mediated endothelial permeability, and transendothelial migration of activated leukocytes were assessed with TUDCA in vitro. Results: Compared with WT mice, tie2-TNF mice showed a decreased visual function correlated with a decrease in protein kinase C α (PKCα) in rod bipolar cells, and increased vascular permeability was further exacerbated in diabetic-tie2-TNF mice. Conversely, TUDCA alleviated these changes in diabetic mice. An increase in inflammation and ER stress in retina coincided with an increase in TGR5 expression in diabetic tie2-TNF mice that decreased with TUDCA. In vitro, HRECs exposed to TNF+HG demonstrated >2-fold increase in TGR5 expression, a 3-fold increase in leukocyte transmigration with a concomitant increase in permeability. Although TUDCA reversed these effects, HRECs silenced for TGR5 and challenged with TUDCA or TGR5 agonist failed to reverse the TNF+HG induced effects. Conclusions: Our data suggest that TUDCA will serve as an excellent therapeutic agent for diabetic complications addressing both vascular and neurodegenerative changes in the retina. Perturbation of the TGR5 receptor in the retina might play a role in linking retinal ER stress to neurovascular dysfunction in diabetic retinopathy.

A Long-Term Safety and Efficacy Report on Intravitreal Delivery of Adipose Stem Cells and Secretome on Visual Deficits After Traumatic Brain Injury.

Purpose: We compared intravitreal injection of human adipose stem cell concentrated conditioned media (ASC-CCM) to injection of live ASCs for their long-term safety and effectiveness against the visual deficits of mild traumatic brain injury (mTBI). Methods: We first tested different intravitreal ASC doses for safety. Other C57BL/6 mice then received focal cranial blast mTBI and were injected with the safe ASC dose (1000 cells/eye), ASC-CCM (∼200 ng protein/eye), or saline solution. At five and 10 months after blast injury, visual, molecular, and histological assessments evaluated treatment efficacy. Histological evaluation of eyes and other organs at 10 months after blast injury assessed safety. Results: Human ASCs at 1000 cells/eye were found to be safe, with >10,000 cells causing retinal damage. Blast-injured mice showed significant vision deficits compared to sham blast mice up to 10 months. Blast mice receiving ASC or ASC-CCM showed improved vision at five months but marginal effects at 10 months, correlated with changes in glial fibrillary acidic protein and proinflammatory gene expression in retina. Immunostaining for human IgG failed to detect ASCs in retina. Peripheral organs examined histologically at 10 months after blast injury were normal. Conclusions: Intravitreal injection of ASCs or ASC-CCM is safe and effective against the visual deficits of mTBI. Considering the unimproved glial response and the risk of retinal damage with live cells, our studies suggest that ASC-CCM has better safety and effectiveness than live cells for the treatment of visual dysfunction in mTBI. Translational Relevance: This study demonstrates the safety and efficacy of mesenchymal stem cell-based therapeutics, supporting them for phase 1 clinical studies.

Mesenchymal stem cell secretome protects against oxidative stress-induced ocular blast visual pathologies.

Visual deficits are a common concern among subjects with head trauma. Stem cell therapies have gained recent attention in treating visual deficits following head trauma. Previously, we have shown that adipose-derived stem cell (ASC) concentrated conditioned medium (ASC-CCM), when delivered via an intravitreal route, yielded a significant improvement in vision accompanied by a decrease in retinal neuroinflammation in a focal cranial blast model that indirectly injures the retina. The purpose of the current study is to extend our previous studies to a direct ocular blast injury model to further establish the preclinical efficacy of ASC-CCM. Adult C57BL/6J mice were subjected to repetitive ocular blast injury (rOBI) of 25 psi to the left eye, followed by intravitreal delivery of ASC-CCM (∼200 ng protein/2 µl) or saline within 2-3 h. Visual function and histological changes were measured 4 weeks after injury and treatment. In vitro, Müller cells were used to evaluate the antioxidant effect of ASC-CCM. Visual acuity, contrast sensitivity, and b-wave amplitudes in rOBI mice receiving saline were significantly decreased compared with age-matched sham blast mice. Immunohistological analyses demonstrated a significant increase in glial fibrillary acidic protein (a retinal injury marker) in Müller cell processes, DNA/RNA damage, and nitrotyrosine (indicative of oxidative stress) in the retina, while qPCR analysis revealed a >2-fold increase in pro-inflammatory cytokines (TNF-α, ICAM1, and Ccl2) in the retina, as well as markers for microglia/macrophage activation (IL-1ß and CD86). Remarkably, rOBI mice that received ASC-CCM demonstrated a significant improvement in visual function compared to saline-treated rOBI mice, with visual acuity, contrast sensitivity, and b-wave amplitudes that were not different from those in sham mice. This improvement in visual function also was associated with a significant reduction in retinal GFAP, neuroinflammation markers, and oxidative stress compared to saline-treated rOBI mice. In vitro, Müller cells exposed to oxidative stress via increasing doses of hydrogen peroxide demonstrated decreased viability, increased GFAP mRNA expression, and reduced activity for the antioxidant catalase. On the other hand, oxidatively stressed Müller cells pre-incubated with ASC-CCM showed normalized GFAP, viability, and catalase activity. In conclusion, our study demonstrates that a single intravitreal injection of ASC-CCM in the rOBI can significantly rescue retinal injury and provide significant restoration of visual function. Our in vitro studies suggest that the antioxidant catalase may play a major role in the protective effects of ASC-CCM, uncovering yet another aspect of the multifaceted benefits of ASC secretome therapies in neurotrauma.

Adipose Tissue-Derived Mesenchymal Stem Cell Concentrated Conditioned Medium Alters the Expression Pattern of Glutamate Regulatory Proteins and Aquaporin-4 in the Retina after Mild Traumatic Brain Injury.

Concentrated conditioned media from adipose tissue-derived mesenchymal stem cells (ASC-CCM) show promise for retinal degenerative diseases. In this study, we hypothesized that ASC-CCM could rescue retinal damage and thereby improve visual function by acting through Müller glia in mild traumatic brain injury (mTBI). Adult C57Bl/6 mice were subjected to a 50-psi air pulse on the left side of the head, resulting in an mTBI. After blast injury, 1 µL (∼100 ng total protein) of human ASC-CCM was delivered intravitreally and followed up after 4 weeks for visual function assessed by electroretinogram and histopathological markers for Müller cell-related markers. Blast mice that received ASC-CCM, compared with blast mice that received saline, demonstrated a significant improvement in a- and b-wave response correlated with a 1.3-fold decrease in extracellular glutamate levels and a concomitant increase in glutamine synthetase (GS), as well as the glutamate transporter (GLAST) in Müller cells. Additionally, an increase in aquaporin-4 (AQP4) in Müller cells in blast mice received saline restored to normal levels in blast mice that received ASC-CCM. In vitro studies on rMC-1 Müller glia exposed to 100 ng/mL glutamate or RNA interference knockdown of GLAST expression mimicked the increased Müller cell glial fibrillary acidic protein (a marker of gliosis) seen with mTBI, and suggested that an increase in glutamate and/or a decrease in GLAST might contribute to the Müller cell activation in vivo. Taken together, our data suggest a novel neuroprotective role for ASC-CCM in the rescue of the visual deficits and pathologies of mTBI via restoration of Müller cell health.

TSG-6 in conditioned media from adipose mesenchymal stem cells protects against visual deficits in mild traumatic brain injury model through neurovascular modulation.

BACKGROUND: Retinal inflammation affecting the neurovascular unit may play a role in the development of visual deficits following mild traumatic brain injury (mTBI). We have shown that concentrated conditioned media from adipose tissue-derived mesenchymal stem cells (ASC-CCM) can limit retinal damage from blast injury and improve visual function. In this study, we addressed the hypothesis that TNFα-stimulated gene-6 (TSG-6), an anti-inflammatory protein released by mesenchymal cells, mediates the observed therapeutic potential of ASCs via neurovascular modulation. METHODS: About 12-week-old C57Bl/6 mice were subjected to 50-psi air pulse on the left side of the head overlying the forebrain resulting in an mTBI. Age-matched sham blast mice served as control. About 1 µl of ASC-CCM (siControl-ASC-CCM) or TSG-6 knockdown ASC-CCM (siTSG-6-ASC-CCM) was delivered intravitreally into both eyes. One month following injection, the ocular function was assessed followed by molecular and immunohistological analysis. In vitro, mouse microglial cells were used to evaluate the anti-inflammatory effect of ASC-CCM. Efficacy of ASC-CCM in normalizing retinal vascular permeability was assessed using trans-endothelial resistance (TER) and VE-cadherin expression in the presence of TNFα (1 ng/ml). RESULTS: We show that intravitreal injection of ASC-CCM (siControl-ASC-CCM) but not the TSG-6 knockdown ASC-CCM (siTSG-6-ASC-CCM) mitigates the loss of visual acuity and contrast sensitivity, retinal expression of genes associated with microglial and endothelial activation, and retinal GFAP immunoreactivity at 4 weeks after blast injury. In vitro, siControl-ASC-CCM but not the siTSG-6-ASC-CCM not only suppressed microglial activation and STAT3 phosphorylation but also protected against TNFα-induced endothelial permeability as measured by transendothelial electrical resistance and decreased STAT3 phosphorylation. CONCLUSIONS: Our findings suggest that ASCs respond to an inflammatory milieu by secreting higher levels of TSG-6 that mediates the resolution of the inflammatory cascade on multiple cell types and correlates with the therapeutic potency of the ASC-CCM. These results expand our understanding of innate mesenchymal cell function and confirm the importance of considering methods to increase the production of key analytes such as TSG-6 if mesenchymal stem cell secretome-derived biologics are to be developed as a treatment solution against the traumatic effects of blast injuries and other neurovascular inflammatory conditions of the retina.

Adipose stem cells and their paracrine factors are therapeutic for early retinal complications of diabetes in the Ins2Akita mouse.

BACKGROUND: Early-stage diabetic retinopathy (DR) is characterized by neurovascular defects. In this study, we hypothesized that human adipose-derived stem cells (ASCs) positive for the pericyte marker CD140b, or their secreted paracrine factors, therapeutically rescue early-stage DR features in an Ins2Akita mouse model. METHODS: Ins2Akita mice at 24 weeks of age received intravitreal injections of CD140b-positive ASCs (1000 cells/1 µL) or 20× conditioned media from cytokine-primed ASCs (ASC-CM, 1 µL). Age-matched wildtype mice that received saline served as controls. Visual function experiments and histological analyses were performed 3 weeks post intravitreal injection. Biochemical and molecular analyses assessed the ASC-CM composition and its biological effects. RESULTS: Three weeks post-injection, Ins2Akita mice that received ASCs had ameliorated decreased b-wave amplitudes and vascular leakage but failed to improve visual acuity, whereas Ins2Akita mice that received ASC-CM demonstrated amelioration of all aforementioned visual deficits. The ASC-CM group demonstrated partial amelioration of retinal GFAP immunoreactivity and DR-related gene expression but the ASC group did not. While Ins2Akita mice that received ASCs exhibited occasional (1 in 8) hemorrhagic retinas, mice that received ASC-CM had no adverse complications. In vitro, ASC-CM protected against TNFα-induced retinal endothelial permeability as measured by transendothelial electrical resistance. Biochemical and molecular analyses demonstrated several anti-inflammatory proteins including TSG-6 being highly expressed in cytokine-primed ASC-CM. CONCLUSIONS: ASCs or their secreted factors mitigate retinal complications of diabetes in the Ins2Akita model. Further investigation is warranted to determine whether ASCs or their secreted factors are safe and effective therapeutic modalities long-term as current locally delivered therapies fail to effectively mitigate the progression of early-stage DR. Nonetheless, our study sheds new light on the therapeutic mechanisms of adult stem cells, with implications for assessing relative risks/benefits of experimental regenerative therapies for vision loss.

Featured Article: Deterioration of visual function mediated by senescence-associated endoplasmic reticulum stress in inflammatory tie2-TNF mice.

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.m2 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.

Concentrated Conditioned Media from Adipose Tissue Derived Mesenchymal Stem Cells Mitigates Visual Deficits and Retinal Inflammation Following Mild Traumatic Brain Injury.

Blast concussions are a common injury sustained in military combat today. Inflammation due to microglial polarization can drive the development of visual defects following blast injuries. In this study, we assessed whether anti-inflammatory factors released by the mesenchymal stem cells derived from adipose tissue (adipose stem cells, ASC) can limit retinal tissue damage and improve visual function in a mouse model of visual deficits following mild traumatic brain injury. We show that intravitreal injection of 1 µL of ASC concentrated conditioned medium from cells pre-stimulated with inflammatory cytokines (ASC-CCM) mitigates loss of visual acuity and contrast sensitivity four weeks post blast injury. Moreover, blast mice showed increased retinal expression of genes associated with microglial activation and inflammation by molecular analyses, retinal glial fibrillary acidic protein (GFAP) immunoreactivity, and increased loss of ganglion cells. Interestingly, blast mice that received ASC-CCM improved in all parameters above. In vitro, ASC-CCM not only suppressed microglial activation but also protected against Tumor necrosis alpha (TNFα) induced endothelial permeability as measured by transendothelial electrical resistance. Biochemical and molecular analyses demonstrate TSG-6 is highly expressed in ASC-CCM from cells pre-stimulated with TNFα and IFNγ but not from unstimulated cells. Our findings suggest that ASC-CCM mitigates visual deficits of the blast injury through their anti-inflammatory properties on activated pro-inflammatory microglia and endothelial cells. A regenerative therapy for immediate delivery at the time of injury may provide a practical and cost-effective solution against the traumatic effects of blast injuries to the retina.