Lisa J Hill

Mesenchymal stromal cell-mediated neuroprotection and functional preservation of retinal ganglion cells in a rodent model of glaucoma.

BACKGROUND AIMS Glaucoma is a leading cause of irreversible blindness involving loss of retinal ganglion cells (RGC). Mesenchymal stromal cells (MSC) have shown promise as a paracrine-mediated therapy for compromised neurons. It is, however, unknown whether dental pulp stem cells (DPSC) are effective as a cellular therapy in glaucoma and how their hypothesized influence compares with other more widely researched MSC sources. The present study aimed to compare the efficacy of adipose-derived stem cells, bone marrow-derived MSC (BMSC) and DPSC in preventing the loss of RGC and visual function when transplanted into the vitreous of glaucomatous rodent eyes. METHODS Thirty-five days after raised intraocular pressure (IOP) and intravitreal stem cell transplantation, Brn3a(+) RGC numbers, retinal nerve fibre layer thickness (RNFL) and RGC function were evaluated by immunohistochemistry, optical coherence tomography and electroretinography, respectively. RESULTS Control glaucomatous eyes that were sham-treated with heat-killed DPSC had a significant loss of RGC numbers, RNFL thickness and function compared with intact eyes. BMSC and, to a greater extent, DPSC provided significant protection from RGC loss and RNFL thinning and preserved RGC function. DISCUSSION The study supports the use of DPSC as a neuroprotective cellular therapy in retinal degenerative disease such as glaucoma.

Nicotinamide, NAD(P)(H), and Methyl-Group Homeostasis Evolved and Became a Determinant of Ageing Diseases: Hypotheses and Lessons from Pellagra

Compartmentalized redox faults are common to ageing diseases. Dietary constituents are catabolized to NAD(H) donating electrons producing proton-based bioenergy in coevolved, cross-species and cross-organ networks. Nicotinamide and NAD deficiency from poor diet or high expenditure causes pellagra, an ageing and dementing disorder with lost robustness to infection and stress. Nicotinamide and stress induce Nicotinamide-N-methyltransferase (NNMT) improving choline retention but consume methyl groups. High NNMT activity is linked to Parkinsons, cancers, and diseases of affluence. Optimising nicotinamide and choline/methyl group availability is important for brain development and increased during our evolution raising metabolic and methylome ceilings through dietary/metabolic symbiotic means but strict energy constraints remain and life-history tradeoffs are the rule. An optimal energy, NAD and methyl group supply, avoiding hypo and hyper-vitaminoses nicotinamide and choline, is important to healthy ageing and avoids utilising double-edged symbionts or uncontrolled autophagy or reversions to fermentation reactions in inflammatory and cancerous tissue that all redistribute NAD(P)(H), but incur high allostatic costs.

Decorin Reduces Intraocular Pressure and Retinal Ganglion Cell Loss in Rodents Through Fibrolysis of the Scarred Trabecular Meshwork

PURPOSE To investigate whether Decorin, a matrikine that regulates extracellular matrix (ECM) deposition, can reverse established trabecular meshwork (TM) fibrosis, lower IOP, and reduce progressive retinal ganglion cell (RGC) death in a novel rodent model of TM fibrosis. METHODS Adult rats had intracameral (IC) injections of human recombinant (hr) TGF-β over 30 days (30 d; to induce TM fibrosis, raise IOP, and initiate RGC death by 17 d) or PBS (controls) and visually evoked potentials (VEP) were measured at 30 d to evaluate resultant visual pathway dysfunction. In some animals TGF-β injections were stopped at 17 d when TM fibrosis and IOP were consistently raised and either hrDecorin or PBS IC injections were administered between 21 d and 30 d. Intraocular pressure was measured biweekly and eyes were processed for immunohistochemical analysis of ECM deposition to assess TM fibrosis and levels of matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) to assess fibrolysis. The effect of hrDecorin treatment on RGC survival was also assessed. RESULTS Transforming growth factor-β injections caused sustained increases in ECM deposition in the TM and raised IOP by 17 d, responses that were associated with 42% RGC loss and a significant decrease in VEP amplitude measured at 30 d. Decorin treatment from 17 d reduced TGF-β-induced TM fibrosis, increased levels of MMP2 and MMP9 and lowered TIMP2 levels, and lowered IOP, preventing progressive RGC loss. CONCLUSIONS Human recombinant Decorin reversed established TM fibrosis and lowered IOP, thereby rescuing RGC from progressive death. These data provide evidence for the candidacy of hrDecorin as a treatment for open-angle glaucoma.

Fusion or fission: The destiny of mitochondria in traumatic brain injury of different severities

Mitochondrial dynamics are regulated by a complex system of proteins representing the mitochondrial quality control (MQC). MQC balances antagonistic forces of fusion and fission determining mitochondrial and cell fates. In several neurological disorders, dysfunctional mitochondria show significant changes in gene and protein expression of the MQC and contribute to the pathophysiological mechanisms of cell damage. In this study, we evaluated the main gene and protein expression involved in the MQC in rats receiving traumatic brain injury (TBI) of different severities. At 6, 24, 48 and 120 hours after mild TBI (mTBI) or severe TBI (sTBI), gene and protein expressions of fusion and fission were measured in brain tissue homogenates. Compared to intact brain controls, results showed that genes and proteins inducing fusion or fission were upregulated and downregulated, respectively, in mTBI, but downregulated and upregulated, respectively, in sTBI. In particular, OPA1, regulating inner membrane dynamics, cristae remodelling, oxidative phosphorylation, was post-translationally cleaved generating differential amounts of long and short OPA1 in mTBI and sTBI. Corroborated by data referring to citrate synthase, these results confirm the transitory (mTBI) or permanent (sTBI) mitochondrial dysfunction, enhancing MQC importance to maintain cell functions and indicating in OPA1 an attractive potential therapeutic target for TBI.

Topical Delivery of Anti-VEGF Drugs to the Ocular Posterior Segment Using Cell-Penetrating Peptides

Purpose To evaluate the efficacy of anti-VEGF agents for treating choroidal neovascularization (CNV) when delivered topically using novel cell-penetrating peptides (CPPs) compared with delivery by intravitreal (ivit) injection. Methods CPP toxicity was investigated in cell cultures. Ivit concentrations of ranibizumab and bevacizumab after topical administration were measured using ELISA. The biological efficacy of topical anti-VEGF + CPP complexes was compared with ivit anti-VEGF injections using an established model of CNV. Results CPPs were nontoxic in vitro. In vivo, after topical eye drop delivery, CPPs were present in the rat anterior chamber within 6 minutes. A single application of CPP + bevacizumab eye drop delivered clinically relevant concentrations of bevacizumab to the posterior chamber of the rat eye in vivo. Similarly, clinically relevant levels of CPP + ranibizumab and CPP + bevacizumab were detected in the porcine vitreous and retina ex vivo. In an established model of CNV, mice treated with either a single ivit injection of anti-VEGF, twice daily CPP + anti-VEGF eye drops or daily dexamethasone gavage for 10 days all had significantly reduced areas of CNV when compared with lasered eyes without treatment. Conclusions CPPs are nontoxic to ocular cells and can be used to deliver therapeutically relevant doses of ranibizumab and bevacizumab by eye drop to the posterior segment of mouse, rat, and pig eyes. The CPP + anti-VEGF drug complexes were cleared from the retina within 24 hours, suggesting a daily eye drop dosing regimen. Daily, topically delivered anti-VEGF with CPP was as efficacious as a single ivit injection of anti-VEGF in reducing areas of CNV in vivo.

Structuring of Hydrogels across Multiple Length Scales for Biomedical Applications

The development of new materials for clinical use is limited by an onerous regulatory framework, which means that taking a completely new material into the clinic can make translation economically unfeasible. One way to get around this issue is to structure materials that are already approved by the regulator, such that they exhibit very distinct physical properties and can be used in a broader range of clinical applications. Here, the focus is on the structuring of soft materials at multiple length scales by modifying processing conditions. By applying shear to newly forming materials, it is possible to trigger molecular reorganization of polymer chains, such that they aggregate to form particles and ribbon-like structures. These structures then weakly interact at zero shear forming a solid-like material. The resulting self-healing network is of particular use for a range of different biomedical applications. How these materials are used to allow the delivery of therapeutic entities (cells and proteins) and as a support for additive layer manufacturing of larger-scale tissue constructs is discussed. This technology enables the development of a range of novel materials and structures for tissue augmentation and regeneration.

Meat Intake and the Dose of Vitamin B3 - Nicotinamide: Cause of the Causes of Disease Transitions, Health Divides, and Health Futures?

Meat and vitamin B3 – nicotinamide – intake was high during hunter-gatherer times. Intake then fell and variances increased during and after the Neolithic agricultural revolution. Health, height, and IQ deteriorated. Low dietary doses are buffered by ‘welcoming’ gut symbionts and tuberculosis that can supply nicotinamide, but this co-evolved homeostatic metagenomic strategy risks dysbioses and impaired resistance to pathogens. Vitamin B3 deficiency may now be common among the poor billions on a low-meat diet. Disease transitions to non-communicable inflammatory disorders (but longer lives) may be driven by positive ‘meat transitions’. High doses of nicotinamide lead to reduced regulatory T cells and immune intolerance. Loss of no longer needed symbiotic ‘old friends’ compounds immunological over-reactivity to cause allergic and auto-immune diseases. Inhibition of nicotinamide adenine dinucleotide consumers and loss of methyl groups or production of toxins may cause cancers, metabolic toxicity, or neurodegeneration. An optimal dosage of vitamin B3 could lead to better health, but such a preventive approach needs more equitable meat distribution. Some people may require personalised doses depending on genetic make-up or, temporarily, when under stress.

Cystatin D (CST5): An ultra-early inflammatory biomarker of traumatic brain injury

Traumatic brain injury (TBI) is set to become the leading cause of neurological disability across all age groups. Currently, no reliable biomarkers exist to help diagnose the severity of TBI to identify patients who are at risk of developing secondary injuries. Thus, the discovery of reliable biomarkers for the management of TBI would improve clinical interventions. Inflammatory markers are particularly suited for biomarker discovery as TBI leads to very early alterations in inflammatory proteins. Using the Proseek Multiplex Inflammation assay, we measured in patients that had suffered mild TBI (n = 10) or severe TBI (n = 10) with extra-cranial injury or extracranial injury only (EC) (n = 10), 92 inflammation-associated proteins in serum obtained: <1 hr (within 1-hour), 4–12 hr and 48–72 hr post injury. Changes were compared to healthy volunteers (HV). Our results identified CST5, AXIN1 and TRAIL as novel early biomarkers of TBI. CST5 identified patients with severe TBI from all other cohorts and importantly was able to do so within the first hour of injury. AXIN1 and TRAIL were able to discriminate between TBI and HV at <1 hr. We conclude that CST5, AXIN1 and TRAIL are worthy of further study in the context of a pre-hospital or pitch-side test to detect brain injury.

Meat and Nicotinamide: A Causal Role in Human Evolution, History, and Demographics:

Hunting for meat was a critical step in all animal and human evolution. A key brain-trophic element in meat is vitamin B3 /nicotinamide. The supply of meat and nicotinamide steadily increased from the Cambrian origin of animal predators ratcheting ever larger brains. This culminated in the 3-million-year evolution of Homo sapiens and our overall demographic success. We view human evolution, recent history, and agricultural and demographic transitions in the light of meat and nicotinamide intake. A biochemical and immunological switch is highlighted that affects fertility in the ‘de novo’ tryptophan-to-kynurenine-nicotinamide ‘immune tolerance’ pathway. Longevity relates to nicotinamide adenine dinucleotide consumer pathways. High meat intake correlates with moderate fertility, high intelligence, good health, and longevity with consequent population stability, whereas low meat/high cereal intake (short of starvation) correlates with high fertility, disease, and population booms and busts. Too high a meat intake and fertility falls below replacement levels. Reducing variances in meat consumption might help stabilise population growth and improve human capital.

Decorin treatment for reversing trabecular meshwork fibrosis in open-angle glaucoma.

In this perspective, we discuss the use of an anti-fibrotic agent Decorin to treat established fibrosis associated with glaucoma originally published by Hill et al. (2015). Glaucoma describes a group of progressive optic neuropathies that have the potential to cause irreversible blindness, for which it is recognized that a main risk factor is raised intraocular pressure (IOP). Currently, there is no precise explanation of why high IOP causes retinal ganglion cell (RGC) death. Factors contributing to IOP-related RGC death include compromised retrograde axonal transport of target derived neurotrophic factors, leading to activation of apoptosis and mitochondrial dysfunction after the biomechanical and ischemic insult of optic nerve head compression. In open-angle glaucoma (OAG), increases in IOP occur when aqueous humour (AqH) outflow through the trabecular meshwork (TM) is reduced, usually as a result of progressive abnormalities in TM cellularity and extracellular matrix (ECM) levels culminating in TM fibrosis (Prendes et al., 2013). These cellular and ECM changes (which includes upregulation of collagen IV, laminin, and fibronectin) in the TM, together with altered TM cell contractility, result in TM dysfunction and ultimately loss of the tightly controlled AqH outflow. Currently, treatments for glaucoma are symptomatic and focus on the use of drugs formulated as eye drops to lower IOP either by reducing AqH production or increasing AqH outflow through non-TM pathways. Alternatively, surgical insertion of shunts through the TM can drain AqH directly to the exterior of the eye, although these often become blocked. For some patients it seems that primary open-angle glaucoma is a fibroproliferative condition and no current treatment addresses the TM fibrosis that is the predominant underlying causes of TM dysregulation. The mechanisms that lead to TM dysfunction in OAG are multifactorial and may relate to dysregulated ocular inflammation since pathologically high levels of the pro-inflammatory cytokine transforming growth factor β (TGF-β) within the AqH and TM have been implicated. For example, some OAG patients have elevated levels of TGF-β1 and/or TGF-β2 in their AqH compared to age-matched patients with other forms of glaucoma (e.g., primary angle closure glaucoma and uveitis-associated secondary glaucoma) and with cataracts (Prendes et al., 2013). Other studies have shown that it is the TGF-β2 isoform that is significantly elevated in eyes with primary OAG compared with non-glaucomatous eyes, and have shown elevated TGF-β1 and TGF-β3 isoforms in pseudoexfoliative glaucoma and in primary angle closure glaucoma (Prendes et al., 2013). TGF-β1 and TGF-β2 are known potent fibrogenic factors and, at pathologically high levels in the eye, can induce the overexpression of ECM proteins that lead to TM dysfunctions (Prendes et al., 2013). TGF-βs also prevent the breakdown of ECM by inhibiting matrix metalloproteinases (MMP) (Ahmed et al. 2014), creating a pro-fibrotic microenvironment within the TM. TGF-β is directly implicated in the pathogenesis of ocular hypertension since: (1) infusion of TGF-β2 for 14 days in an anterior eye segment perfusion culture model significantly increased IOP (Battacharya et al., 2009); (2) adenoviral gene delivery of human TGF-β2 into the anterior segment to the rodent eye reduced AqH outflow and increased IOP (Shepard et al., 2010); and (3) TGF-β1 gene delivery by adenovirus to the anterior segment of the rat eye increased IOP (Robertson et al., 2010). Thus, treatment of the TM in OAG with a TGF-β antagonist may help to ablate TM fibrogenesis, thereby perturbing the progression of OAG pathology. The matrikine Decorin is a small leucine-rich proteoglycan that regulates cell proliferation, survival and differentiation by antagonizing a panel of growth factors and/or their receptors, including TGF-β, epidermal growth factor, vascular endothelial growth factor, hepatocyte growth factor and insulin-like growth factor-1. Decorin also ‘decorates’ collagen, interfering with collagen fibrillogenesis and stabilizing collagen fibres. In addition, Decorin enhances matrix metalloproteinases (MMP) activity by increasing levels of tissue plasminogen activator (tPA), thus enabling plasmin-dependent activation of MMP by cleavage of plasminogen to plasmin, and reducing levels of PAI-1 and tissue inhibitors of MMPs, further facilitating MMP activation. The fibrolytic actions of Decorin have been harnessed to attenuate the progression of tissue damage in other fibroproliferative pathologies, including juvenile communicating hydrocephalus (Botfield et al., 2013) and spinal cord injury (Ahmed et al., 2014), making Decorin an ideal candidate for treating TM fibrosis and progressive RGC loss in glaucoma. Modelling raised IOP in OAG using intracameral (IC) injections of TGF-β: In the study by Hill et al. (2015), we used immunohistochemistry to visualize fibrosis and count RGCs, tonometry to measure IOP and visual evoked potentials (VEP) to measure function of the visual pathway, and found that repeated (twice a week) intracameral injections of TGF-β in adult rats promoted TM fibrosis, elevated IOP and led to RGC death and retinal dysfunction. Consistent with other fibrotic models of raised IOP, our rodent model generated a sustained and significant increase in IOP by 14 days compared with PBS controls. The baseline level of IOP (before any treatment) in our study was similar to that observed previously in rats (Robertson et al. 2010). However, overexpression of TGF-β by gene transfer (Robertson et al. 2010) led to greater levels of IOP (20 mmHg) compared to IOP levels (14 mmHg) achieved in our study, differences that maybe explained by the constant production of TGF-β through gene transcription compared with our discontinuous bolus regime of biweekly IC injections. One feature of our method was that TGF-β treatment for 17 days led to TM fibrosis and elevated IOP that persisted for at least 30 days despite withdrawal of TGF-β from this time point. To confirm that there was no natural resolution of the TM fibrosis and raised IOP (due to continued ECM turnover in the TM) after the cessation of TGF-β treatment we used a control group in which intracameral injection of TGF-β injections for 17 days was followed by intracameral injection of PBS from 21–30 days with no resolution of IOP. Thus, in this in vivo model of TGF-β-induced TM fibrosis, IOP was increased by 17 days and sustained through to 30 days, resulting in 42% RGC death and associated functional VEP deficits, consequences that were similar to those seen in other experimental models of raised IOP (Belforte et al., 2010). Of note, in the study of Hill et al. (2015) it was important to identify and account for any inflammatory reactions induced by the intracameral injections that may have perpetuated TM fibrosis. This was addressed by counting the number of ED1+ macrophages situated within the proximity of the TM. The results showed that intracameral TGF-β and PBS did increase the number of macrophages in the angle compared with numbers in the intact control eyes. These results suggest that, since there were similar macrophage numbers in the PBS and TGF-β-treated groups, the induced differences between the two treatments were not a consequence of post-surgical inflammation. Evaluating the utility of human recombinant Decorin hrDecorin for the treatment of OAG: Once the model was successfully established, hrDecorin was injected intracamerally after TM fibrosis was established to ascertain if the induced TM fibrosis and increased IOP could be resolved. hrDecorin significantly reduced the laminin and fibronectin levels within the fibrosed TM, an effect that was correlated with raised levels of MMP and their TIMP inhibitors. Thus, hrDecorin reversed the TM microenvironment back to MMP/TIMP ratios favourable for tissue remodelling, causing the dissolution of established ECM protein deposits in and around the TM, an anti-scarring response reminiscent of that seen to Decorin in models of spinal cord injury (Ahmed et al., 2013), hydrocephalus (Botfield et al., 2013) and conjunctival scarring for post-filtration surgery (Grisanti et al., 2005). The study of Hill et al. (2015) in the OAG model also showed that the anti-scarring Decorin treatment lowered IOP when compared with controls in vivo, with associated enhancement of RGC survival. Primary retinal cultures containing RGC demonstrated that the neuroprotective effects of Decorin were indirectly related to the resolution of TM fibrosis and lowering of IOP rather than by direct actions on neurons. However, other studies have shown contradictory results on other cell types. For example, Seidler et al. (2006) reported an anti-apoptotic effect of Decorin on cultured fibroblasts by prevention of DNA fragmentation. By contrast, studies using cancer cells have convincingly demonstrated the ability of Decorin to induce cell death through apoptosis (Goldoni et al., 2008). Therefore, we suggest that the actions of Decorin are dependent on the cell type and their environment. Conclusion: Our observations show that intracameral hrDecorin reverses established TM fibrosis and normalizes IOP, indirectly protecting RGC from progressive IOP-related death. Further confirmatory studies will be required to understand the exact mechanism of Decorins fibrolytic actions within the TM but we believe that hrDecorin is an ideal candidate therapy to develop into a treatment for patients with OAG associated with TM fibrosis. LJH was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), No. BB/F017553/1.