MicroRNA-409-3p/BTG2 signaling axis improves impaired angiogenesis and wound healing in obese mice.

Wound healing is facilitated by neoangiogenesis, a complex process that is essential to tissue repair in response to injury. MicroRNAs are small, noncoding RNAs that can regulate the wound healing process including stimulation of impaired angiogenesis that is associated with type-2 diabetes (T2D). Expression of miR-409-3p was significantly increased in the nonhealing skin wounds of patients with T2D compared to the non-wounded normal skin, and in the skin of a murine model with T2D. In response to high glucose, neutralization of miR-409-3p markedly improved EC growth and migration in human umbilical vein endothelial cells (HUVECs), promoted wound closure and angiogenesis as measured by increased CD31 in human skin organoids, while overexpression attenuated EC angiogenic responses. Bulk mRNA-Seq transcriptomic profiling revealed BTG2 as a target of miR-409-3p, where overexpression of miR-409-3p significantly decreased BTG2 mRNA and protein expression. A 3' untranslated region (3'-UTR) luciferase assay of BTG2 revealed decreased luciferase activity with overexpression of miR-409-3p, while inhibition had opposite effects. Mechanistically, in response to high glucose, miR-409-3p deficiency in ECs resulted in increased mTOR phosphorylation, meanwhile BTG-anti-proliferation factor 2 (BTG2) silencing significantly decreased mTOR phosphorylation. Endothelial-specific and tamoxifen-inducible miR-409-3p knockout mice (MiR-409IndECKO ) with hyperglycemia that underwent dorsal skin wounding showed significant improvement of wound closure, increased blood flow, granulation tissue thickness (GTT), and CD31 that correlated with increased BTG2 expression. Taken together, our results show that miR-409-3p is a critical mediator of impaired angiogenesis in diabetic skin wound healing.

Human red blood cells release microvesicles with distinct sizes and protein composition that alter neutrophil phagocytosis.

Extracellular vesicles (EVs) are membrane-bound structures released by cells and tissues into biofluids, involved in cell-cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell-type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities, and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC-derived microvesicles (RBC-MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis, and vesicular transport, such as of sorcin, stomatin, annexin A7, and RAB proteins. Cryo-electron microscopy identified two separate pathways of RBC-MV-neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC-MVs decrease neutrophil ability to phagocytose E. coli but do not affect their survival at 24 hrs. This work brings new insights regarding the complexity of the RBC-MVs biogenesis, as well as their possible role in circulation.

Immunological consequences of compromised ocular immune privilege accelerate retinal degeneration in retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) is a hereditary retinal disease which leads to visual impairment. The onset and progression of RP has physiological consequences that affects the ocular environment. Some of the key non-genetic factors which hasten the retinal degeneration in RP include oxidative stress, hypoxia and ocular inflammation. In this study, we investigated the status of the ocular immune privilege during retinal degeneration and the effect of ocular immune changes on the peripheral immune system in RP. We assessed the peripheral blood mononuclear cell stimulation by retinal antigens and their immune response status in RP patients. Subsequently, we examined alterations in ocular immune privilege machineries which may contribute to ocular inflammation and disease progression in rd1 mouse model. RESULTS: In RP patients, we observed a suppressed anti-inflammatory response to self-retinal antigens, thereby indicating a deviated response to self-antigens. The ocular milieu in rd1 mouse model indicated a significant decrease in immune suppressive ligands and cytokine TGF-B1, and higher pro-inflammatory ocular protein levels. Further, blood-retinal-barrier breakdown due to decrease in the expression of tight junction proteins was observed. The retinal breach potentiated pro-inflammatory peripheral immune activation against retinal antigens and caused infiltration of the peripheral immune cells into the ocular tissue. CONCLUSIONS: Our studies with RP patients and rd1 mouse model suggest that immunological consequences in RP is a contributing factor in the progression of retinal degeneration. The ocular inflammation in the RP alters the ocular immune privilege mechanisms and peripheral immune response. These aberrations in turn create an auto-reactive immune environment and accelerate retinal degeneration.

A nanoparticle probe for the imaging of autophagic flux in live mice via magnetic resonance and near-infrared fluorescence.

Autophagy-the lysosomal degradation of cytoplasmic components via their sequestration into double-membraned autophagosomes-has not been detected non-invasively. Here we show that the flux of autophagosomes can be measured via magnetic resonance imaging or serial near-infrared fluorescence imaging of intravenously injected iron oxide nanoparticles decorated with cathepsin-cleavable arginine-rich peptides functionalized with the near-infrared fluorochrome Cy5.5 (the peptides facilitate the uptake of the nanoparticles by early autophagosomes, and are then cleaved by cathepsins in lysosomes). In the heart tissue of live mice, the nanoparticles enabled quantitative measurements of changes in autophagic flux, upregulated genetically, by ischaemia-reperfusion injury or via starvation, or inhibited via the administration of a chemotherapeutic or the antibiotic bafilomycin. In mice receiving doxorubicin, pre-starvation improved cardiac function and overall survival, suggesting that bursts of increased autophagic flux may have cardioprotective effects during chemotherapy. Autophagy-detecting nanoparticle probes may facilitate the further understanding of the roles of autophagy in disease.

TRACE-seq: A transgenic system for unbiased and non-invasive transcriptome profiling of living cells.

Dynamic profiling of changes in gene expression in response to stressors in specific microenvironments without requiring cellular destruction remains challenging. Current methodologies that seek to interrogate gene expression at a molecular level require sampling of cellular transcriptome and therefore lysis of the cell, preventing serial analysis of cellular transcriptome. To address this area of unmet need, we have recently developed a technology allowing transcriptomic analysis over time without cellular destruction. Our method, TRACE-seq (TRanscriptomic Analysis Captured in Extracellular vesicles using sequencing), is characterized by a cell-type specific transgene expression. It provides data on the transcriptome inside extracellular vesicles that provides an accurate representation of stress-responsive cellular transcriptomic changes. Thus, the transcriptome of cells expressing TRACE can be followed over time without destroying the source cell, which is a powerful tool for many fields of fundamental and translational biology research.

The safety and efficacy of BCG encapsulated alginate particle (BEAP) against M.tb H37Rv infection in Macaca mulatta : A pilot study.

Due to the limited utility of Bacillus Calmette-Guérin (BCG), the only approved vaccine available for tuberculosis, there is a need to develop a more effective and safe vaccine. We evaluated the safety and efficacy of a dry powder aerosol (DPA) formulation of BCG encapsulated alginate particle (BEAP) and the conventional intradermal BCG immunization in infant rhesus macaques (Macaca mulatta). The infant macaques were immunized intratracheally with DPA of BEAP into the lungs. Animals were monitored for their growth, behaviour, any adverse and allergic response. The protective efficacy of BEAP was estimated by the ex-vivo H37Rv infection method. Post-immunization with BEAP, granulocytes count, weight gain, chest radiography, levels of liver secreted enzymes, cytokines associated with inflammation like TNF and IL-6 established that BEAP is non-toxic and it does not elicit an allergic response. The T cells isolated from BEAP immunized animals' blood, upon stimulation with M.tb antigen, secreted high levels of IFN-γ, TNF, IL-6 and IL-2. The activated T cells from BEAP group, when co-cultured with M.tb infected macrophages, eliminated largest number of infected macrophages compared to the BCG and control group. This study suggests the safety and efficacy of BEAP in Non-human primate model.

Apoptosis-inducing factor deficient mice fail to develop hepatic steatosis under high fat high fructose diet or bile duct ligation.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein involved in redox signalling and programmed cell death. The role of AIF has been well recognized in diabetes and obesity. However, the aspect of AIF deficiency in the development of hepatic steatosis and liver injury is unknown. Therefore, in the current study, Harlequin (Hq mutant) mouse with markedly reduced content of AIF was investigated to explore the role of AIF on the initiation of liver injury. The wild type (WT) developed physiological and pathological features of non-alcoholic fatty liver disease (NAFLD) that were not seen in the Hq mice with AIF deficiency, when fed on high fat high fructose (HFHF) diet. Following bile duct ligation (BDL), the liver associated pathological changes were less conspicuous in Hq mice as compared to WT mice. The expression of AIF protein and apoptosis was markedly lesser as compared to their respective control in Hq mice on HFHF diet. Furthermore, the genes involved in fatty acid metabolism were also altered in the group of treated Hq mice. In conclusion, Hq mice failed to develop diet induced hepatic steatosis, suggestive of a role of AIF mediated pathway in the initiation and progression of liver inflammation. Thus, partial loss of AIF appears to be hepatoprotective. SIGNIFICANCE OF THE STUDY: AIF deficiency has multiple roles in altered pathology processes and cellular metabolism, thereby compromising the cellular homeostasis. Considering the molecular functions of AIF in other organ pathology little is known about its role in diet induced liver injury. Hence, the aim of the current study was to investigate the role of AIF deficiency in liver injury and diseases with focus on NAFLD. The study will help to deliniate the mechanisms of NAFLD using Harliquin Mice.

Peripheral blood-derived monocytes show neuronal properties and integration in immune-deficient rd1 mouse model upon phenotypic differentiation and induction with retinal growth factors.

BACKGROUND: Cell therapy is one of the most promising therapeutic interventions for retinitis pigmentosa. In the current study, we aimed to assess if peripheral blood-derived monocytes which are highly abundant and accessible could be utilized as a potential candidate for phenotypic differentiation into neuron-like cells. METHODS: The peripheral blood-derived monocytes were reconditioned phenotypically using extrinsic growth factors to induce pluripotency and proliferation. The reconditioned monocytes (RM) were further incubated with a cocktail of growth factors involved in retinal development and growth to induce retinal neuron-like properties. These cells, termed as retinal neuron-like cells (RNLCs) were characterized for their morphological, molecular and functional behaviour in vitro and in vivo. RESULTS: The monocytes de-differentiated in vitro and acquired pluripotency with the expression of prominent stem cell markers. Treatment of RM with retinal growth factors led to an upregulation of neuronal and retinal lineage markers and downregulation of myeloid markers. These cells show morphological alterations resembling retinal neuron-like cells and expressed photoreceptor (PR) markers. The induced RNLCs also exhibited relative membrane potential change upon light exposure suggesting that they have gained some neuronal characteristics. Further studies showed that RNLCs could also integrate in an immune-deficient retinitis pigmentosa mouse model NOD.SCID-rd1 upon sub-retinal transplantation. The RNLCs engrafted in the inner nuclear layer (INL) and ganglion cell layer (GCL) of the RP afflicted retina. Mice transplanted with RNLCs showed improvement in depth perception, exploratory behaviour and the optokinetic response. CONCLUSIONS: This proof-of-concept study demonstrates that reconditioned monocytes can be induced to acquire retinal neuron-like properties through differentiation using a defined growth media and can be a potential candidate for cell therapy-based interventions and disease modelling for ocular diseases.

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen.

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

Aerosol immunization by alginate coated mycobacterium (BCG/MIP) particles provide enhanced immune response and protective efficacy than aerosol of plain mycobacterium against M.tb. H37Rv infection in mice.

BACKGROUND: With the aim of preparing a more effective, safe and economical vaccine for tuberculosis, inhalable live mycobacterium formulations were evaluated. METHODS: Alginate particles in the size range of 2-4 µm were prepared by encapsulating live Bacille Calmette-Guérin (BCG) and "Mycobacterium indicus pranii" (MIP). These particles were characterized for their size, stability and release profile. Mice were immunized with liquid aerosol or dry powder aerosol (DPA) alginate encapsulated mycobacterium particles and their in-vitro recall response and infection with mycobacterium H37Rv were investigated. RESULTS: It was found that the DPA of alginate encapsulated mycobacterium particles invoked superior immune response and provided higher protection in mice than the liquid aerosol. The BCG encapsulated in alginate particles (BEAP) and MIP encapsulated in alginate particles (MEAP) were engulfed by bone marrow dendritic cells (BMDCs) and co-localized with lysosome. The MEAP/BEAP activated BMDCs exhibited higher chemotaxis movement and had enhanced ability of antigen presentation to T cells. The in-vitro recall response of BEAP/MEAP immunized mice when compared in terms of proliferation index and Interferon gamma (IFN-gamma) released by splenocytes and mediastinal lymph node cells was found to be higher than mice immunized by liquid aerosol of BCG/MIP. Finally, different groups of immunized mice were infected with M. tb H37Rv and after 16 weeks the Colony forming units (CFUs) in lung and spleen estimated. The bacilli burden in the BEAP/MEAP immunized mice was significantly less than the respective liquid aerosol immunized mice and the histopathology of BEAP/MEAP immunized mice lungs showed very little damage. CONCLUSIONS: These inhale-able vaccines formulation of alginate coated live mycobacterium are more immunogenic as compared to the aerosol of bacilli and they provide better protection in mice when infected with H37Rv.

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice.

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

A novel immunodeficient NOD.SCID-rd1 mouse model of retinitis pigmentosa to investigate potential therapeutics and pathogenesis of retinal degeneration.

Retinitis pigmentosa (RP) is a common retinal degeneration disease caused by mutation in any gene of the photo transduction cascade and results in photoreceptor dystrophy. Over decades, several animal models have been used to address the need for the elucidation of effective therapeutics and factors regulating retinal degeneration to prohibit or renew the damaged retina. However, controversies over the immune privilege of retina during cell transplantation and the role of immune modulation during RP still remain largely uninvestigated because of the lack of suitable animal models. Here, we have developed an immunocompromised mouse model, NOD.SCID-rd1, for retinitis pigmentosa (RP) by crossing CBA/J and NOD SCID mice and selecting homozygous double mutant animals for further breeding. Characterization of the newly developed RP model indicates a similar retinal degeneration pattern as CBA/J, with a decreased apoptosis rate and rhodopsin loss. It also exhibits loss of T cells, B cells and NK cells. The NOD.SCID-rd1 model is extremely useful for allogenic and xenogenic cell-based therapeutics, as indicated by the higher cell integration capacity post transplantation. We dissect the underlying role of the immune system in the progression of RP and the effect of immune deficiency on immune privilege of the eye using comparative qPCR studies of this model and the immune-competent RP model.