Citations

Abstract BACKGROUND Diffuse midline gliomas harboring the H3K27M mutation are aggressive and universally fatal brain tumors that primarily occur in children. The blood-brain barrier (BBB) prevents many drugs from reaching the tumor at cytotoxic concentrations and efflux pumps found on the epithelial cells of the BBB rapidly pump drugs out of the brain. Convection enhanced delivery (CED) is a drug delivery technique that bypasses the BBB by directly injecting the drug into the tumor site under a pressure gradient. Further clinical implementation of CED requires understanding drug distribution, as optimal drug physico-chemical properties have never been evaluated. METHODS Sprague Dawley rats underwent a single injection of drug by CED to the brainstem with and without an efflux pump inhibitor. Animals were euthanized at 0, 2, 4, 8, 12 and 24 hours. Their brain drug concentration/distribution was analyzed by MALDI-MSI and plasma drug concentration was measured by LC-MS/MS. RESULTS Drug distribution and brainstem concentration were increased following BBB efflux pump inhibition compared to no pump inhibition controls. Additionally, efflux pump inhibition resulted in slower drug clearance for those drugs that are known pump substrates. CONCLUSIONS BBB efflux pump inhibition resulted in a larger volume of distribution, increased drug concentration and slower drug clearance following CED to the brainstem of known efflux substrates.

Abstract Diffuse midline gliomas (DMG) patients have a dire prognosis despite radiation therapy and there is an urgent need to develop more effective treatments. DMG are characterized by heterozygous mutations in select H3 genes resulting in the replacement of lysine 27 by methionine (K27M) that leads to global epigenetic reprogramming and drives tumorigenesis. We previously reported that pharmacological inhibition of aurora kinase (AKI) may represent a targeted approach for treating tumors with this mutation. Our analysis with both published dataset and patient samples showed that patients with higher aurora kinase A (AKA) expression were associated with worse survival. AKA phosphorylates H3S10 and H3S28 during mitosis. Intriguingly, phosphorylation of the H3S28 (H3S28ph) by AKA blocks PRC2 methyltransferase activity and decreases global H3K27me3 in certain stem cells. We propose that a similar mechanism occurs in H3K27M DMG tumors, where there is a reciprocal relationship between H3S28ph and H3K27me3. We found that AKI significantly decreases H3S28ph while increasing H3K27me3 specifically in H3K27M tumors. To further evaluate the link between the H3K27M mutation and H3 serine phosphorylation, we used CRISPR/Cas9-directed gene editing to silence H3S28ph by replacing serine with alanine (H3S28A) in DIPG cell lines. Ectopic expression of histone H3S28A leads to a prominent epigenetic changes in H3K27M tumors and is similar to AKA inhibition. Overall, this study highlights H3S28ph, one of the targets of AK, is a key driver of epigenetic changes in H3K27M tumors through both direct and indirect changes to H3K27me3 and H3K27ac across the genome.

Abstract Diffuse midline glioma (DMG) is a lethal brain tumor that typically occurs in children. Numerous studies have demonstrated the central role of the H3K27M mutation and secondary loss of H3K27 trimethylation (H3K27me3) in DMG tumorigenesis. Understanding how the H3K27M mutation alters the epigenetic landscape of the cell is necessary for revealing molecular targets that are critical to tumorigenesis. To investigate the epigenetic effects of H3K27M mutation in DMG, we developed revertant DMG cell lines with the mutant methionine residue reverted to wildtype (i.e., M27K). Revertant cells were analyzed for epigenetic changes and phenotypic differences in vitro and in vivo. H3M27K DMG cells grew in culture but displayed diminished proliferative capacity. H3M27K cells demonstrated total loss of H3K27M expression and restored trimethylation of H3K27 and H3K4. Furthermore, consistent with the hypothesis that the H3K27M mutation impacts H3 phosphorylation via expression of Aurora Kinase during mitosis, H3M27K cells demonstrated reduced expression of both Aurora Kinase A and phosphorylation of H3 serine residues 10 and 28. In line with the critical role of H3S10 phosphorylation in chromatin segregation, H3M27K cells also demonstrated restored chromosome segregation compared to H3K27M cells. In vivo data will be discussed. Revertance of the H3K27M mutation reduces tumorigenesis in DMG tumors. Isogenic H3M27K cells display reversal of key epigenetic changes associated with oncogenesis in DMG. The revertant H3M27K DMG model is a useful tool to investigate the downstream epigenetic reprogramming specific to H3K27M mutation in these tumors.

Abstract Background and Aims Novel therapies are needed to address the increasing prevalence of chronic kidney disease. Mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (EVs) augment tissue repair. EVs have practical benefits, but their efficacy relative to MSCs is incompletely understood. We tested the hypothesis that EVs are as effective as MSCs in protecting the stenotic kidney but target different injury pathways. Method Pigs were studied after 16 weeks of renal injury achieved by diet-induced metabolic syndrome (Mets) and renal artery stenosis (RAS). Pigs were untreated or treated four weeks earlier with a single intrarenal delivery of autologous adipose tissue-derived MSCs (1 × 107) or EVs (1 × 1010). Lean pigs and sham RAS served as controls (n=6 each). Stenotic kidney function was studied in-vivo using CT. Histopathology and expression of necroptosis markers (RIPK-1 and RIPK-3), inflammatory and growth factors (angiopoeitin-1 and VEGF) was studied ex-vivo. Results Stenotic-kidney glomerular filtration rate and blood flow in Mets+RAS were both lower than Mets, increased in Mets+RAS+MSCs, and further improved in Mets+RAS+EV. Both MSCs and EVs improved renal function, hypoxia, and decreased renal fibrosis and apoptosis. MSC were slightly more effective in preserving microvascular density (0.02-0.2mm in diameter), and prominently attenuated renal inflammation. However, EVs more significantly upregulated growth-factor expression and decreased necropotosis. Conclusion: Adipose tissue-derived MSCs and their EVs both improve stenotic kidney function and decrease tissue injury in Mets+RAS. MSCs more effectively preserve the microcirculation, while EVs bestow better preservation of renal cellular integrity. These findings encourage further exploration of this novel approach to attenuate renal injury.

Abstract Background and Aims Endothelial progenitor cells (EPCs) patrol the circulation and contribute to endothelial cell regeneration and tissue revascularization. Atherosclerotic renal artery stenosis (ARAS) induces microvascular loss in the stenotic kidney. A regimen of low–energy shockwave therapy (SW) induces angiogenesis and reduces chronic ischemia, but the mechanism remains unclear. This study tested the hypothesis that SW increases EPCs homing to the stenotic kidney and increases renal capillary regeneration in a unilateral ARAS swine model. Method Domestic pigs were randomized to normal control or unilateral ARAS (induced by a local irritant coil in the renal artery and a high-fat diet). ARAS pigs were treated with low-energy SW (0.1 mJ/mm2) or sham (n=6 each group), bi-weekly for 3 consecutive weeks, starting after 3 weeks of ARAS. Blood samples for EPCs (CD34+ and KDR+ by flow cytometry) and the homing factor SDF-1 were collected 4 weeks after completion of SW treatment from the inferior vena cava (IVC) and the stenotic kidney (STK) vein and artery. Urine was collected from the urinary bladder. Kidneys were studied ex vivo for morphology and expression of an endothelial cell marker (CD31) and the pro- angiogenic growth factor angiopoietin (Ang)-1. Results In ARAS, tubulointerstitial fibrosis, tubular score, urinary protein, serum creatinine and mean arterial pressure were significantly increased and capillary count (Fig. 1A) decreased (P<0.05 vs. control), but all markedly improved in ARAS+SW (P<0.05 vs. ARAS). EPC number was decreased in the IVC and renal artery of ARAS pigs (Fig. 1B), but improved in ARAS+SW. SDF-1 levels in the IVC, STK vein and artery, and Ang-1 expression in the kidney of ARAS+SW, were all increased (P<0.05 vs. ARAS). Conclusion Low-energy shockwave improves ischemic kidney capillary density, which is associated with and may be at least in part mediated by promoting EPC mobilization and homing to the stenotic kidney.

Abstract Background and Aims Tumor necrosis factor-α-induced gene/protein (TSG)-6 is a key factor influencing mesenchymal stem cells (MSCs) immunomodulatory properties, but its renoprotective efficacy is unknown. Using a novel swine model of renal artery stenosis (RAS) complicated by metabolic syndrome (Mets), we assessed the therapeutic effects of adipose tissue-derived MSCs-produced TSG-6 and mechanisms underlying the immunomodulatory properties of MSCs. Method Five groups of pigs (n=6 each) were studied after 16 weeks of diet-induced Mets and unilateral RAS (Mets+RAS), either untreated or treated 4 weeks earlier with a single intra-renal delivery of autologous posrcine adipose tissue-derived MSCs (pMSC). Lean, Mets, and RAS shams served as controls. We studied renal function in vivo (using CT imaging) and kidney histopathology and macrophage phenotype ex vivo. In vitro, TSG-6 levels were measured in conditioned media of human MSCs (hMSCs) incubated with or without TNF-α. Additionally, levels of the tubular injury marker LDH were measured in conditioned media after co-culturing macrophages with injured HK-2 cells (achieved by TNF-α and antimycin-A, AMA) with or without addition of TSG-6. The effects of TSG-6 on macrophage phenotype (M1/M2), adhesion, and migration capability were determined. Results Mets+RAS pigs showed increased renal M1 macrophages and renal vein TNF-α levels. After p-MSCs delivery, renal vein TSG-6 increased and TNF-α decreased, M1 macrophage switched to M2 (Fig. A),, renal function improved, and fibrosis alleviated. In vitro, TNF-α increased TSG-6 secretion by h-MSCs. TSG-6 decreased LDH release from injured HK-2, induced a macrophage phenotypic switch from M1 to M2 (Fig. B), and reduced M1 macrophage adhesion and migration (Fig. C). Conclusion TNF-α-induced TSG-6 release from MSCs in vivo and in vitro may decrease renal tubular cells injury, which is associated with and may be at least in part mediated by regulating macrophage function and phenotype.

OBJECTIVE Convection-enhanced delivery (CED) and osmotic pump delivery both have been promoted as promising techniques to deliver drugs to pediatric diffuse intrinsic pontine gliomas (DIPGs). Correspondingly, the aim of this study was to understand how infusate molecular weight (MW), duration of delivery, and mechanism of delivery (CED or osmotic pump) affect volume of distribution (V d ) in the brainstem, to better inform drug selection and delivery in future DIPG investigations. METHODS A series of in vivo experiments were conducted using rat models. CED and osmotic pump delivery systems were surgically implanted in the brainstem, and different MW fluorescent dextran beads were infused either once (acute) or daily for 5 days (chronic) in a volume infused (V i ). Brainstems were harvested after the last infusion, and V d was quantified using serial sectioning and fluorescence imaging. RESULTS Fluorescence imaging showed infusate uptake within the brainstem for both systems without complication. A significant inverse relationship was observed between infusate MW and V d in all settings, which was distinctly exponential in nature in the setting of acute delivery across the 570-Da to 150-kDa range. Chronic duration and CED technique resulted in significantly greater V d compared to acute duration or osmotic pump delivery, respectively. When accounting for V i , acute infusion yielded significantly greater V d /V i than chronic infusion. The distribution in CED versus osmotic pump delivery was significantly affected by infusate MW at higher weights. CONCLUSIONS Here the authors demonstrate that infusate MW, duration of infusion, and infusion mechanism all impact the V d of an infused agent and should be considered when selecting drugs and infusion parameters for novel investigations to treat DIPGs.

Abstract Diffuse midline gliomas harboring the H3K27M mutation, previously known as diffuse intrinsic pontine gliomas (DIPG), are rare and aggressive pediatric brain tumors without cure. One of the major challenge sin DIPG treatment is the effective delivery of therapeutic agents across the blood-brain barrier (BBB) to the tumor and surrounding infiltrating cells. Therefore, strategies that enhance drug delivery to the brain are of great interest. Convection-enhanced delivery (CED) is a technique that bypasses the BBB and increases drug distribution by applying hydraulic pressure to deliver compounds directly and evenly into a target region. However, knowledge in CED pharmacology and convective kinetics is still lacking. In an effort to characterize the feasibility, safety, and distribution in the brain based on molecular size of the delivered agent, we performed infusions of FITC-dextran (range 3,000 Da–150,000 Da) comparing CED and osmotic pump-based delivery into the brainstem of rodents. We calculated the area and volume of distribution (Vd) of the FITC-dextran throughout the brain. Our data showed that the Vd decreased exponentially with increased molecular weight of the FITC-dextran. Interestingly, the Vdcan maintain linearity at lower molecular weights. Maximal cross-sectional area and craniocaudal extension of fluorescence also decreased when lowering the infusate size. In addition, we developed a patient-derived DIPG orthotopic xenograft model and performed an image-guided CED cannula installation in the tumor bed. Using 3D bioluminescence imaging and computed tomography, we determined the tumor volume and the positioning of the cannula in the bulk tumor. The summation of these results supports CED as a promising technique for treating DIPG tumors. A better understanding of how drugs distribute by convection will allow us to optimize treatment regimens and, ultimately, offers hope to patients and families with this devastating disease.

Abstract BACKGROUND Diffuse midline gliomas harboring the H3K27M mutation, previously known as diffuse intrinsic pontine gliomas (DIPG), are rare and aggressive pediatric brain tumors. Over 100 clinical trials with different chemotherapeutics have failed to show any therapeutic benefit. One reason for failure is likely due to poor delivery of these agents to the brainstem. Convection-enhanced delivery (CED) is an emerging delivery technique used to directly inject the agent of interest into the brainstem under pressure. While there is evidence that this may be an effective delivery method, little work has been done to understand the optimal physical properties of these drugs. We sought characterize volume of distribution in the brain based on molecular size of the agent delivered via CED. METHODS Sprague- Dawley rats underwent a single injection of FITC-dextran (3,000 Da, 10,000 Da, 20,000 Da, 70,000 Da, 150,000 Da) via CED into the pons. Post-injection, animals were sacrificed and their brains harvested. Fluorescent microscopy imaging was used to calculate the volume of distribution of the FITC-dextran throughout the brain. RESULTS The volume of distribution (Vd) decreased exponentially according to a two-phase delay (r2= 0.94) as the molecular size of the FITC-dextran increased. The highest mean Vd (107.87mm3) was at a molecular weight of 3,000 Da, and lowest mean Vd (26.48 mm3) was at a molecular weight of 150,000 Da. ANOVA analysis was statistically significant (p= 0.0017). CONCLUSIONS As the molecular size of the FITC-dextran increased, the volume of distribution within the brain following a single injection via CED into the pons decreased. A better understanding of how drugs distribute by convection will allow us to optimize treatment regimens for DIPG tumors.