Enzymatic depletion of circulating glutamine is immunosuppressive in cancers.

Although glutamine addiction in cancer cells is extensively reported, there is controversy on the impact of glutamine metabolism on the immune cells within the tumor microenvironment (TME). To address the role of extracellular glutamine, we enzymatically depleted circulating glutamine using PEGylated Helicobacter pylori gamma-glutamyl transferase (PEG-GGT) in syngeneic mouse models of breast and colon cancers. PEG-GGT treatment inhibits growth of cancer cells in vitro, but in vivo it increases myeloid-derived suppressor cells (MDSCs) and has no significant impact on tumor growth. By deriving a glutamine depletion signature, we analyze diverse human cancers within the TCGA and illustrate that glutamine depletion is not associated with favorable clinical outcomes and correlates with accumulation of MDSC. Broadly, our results help clarify the integrated impact of glutamine depletion within the TME and advance PEG-GGT as an enzymatic tool for the systemic and selective depletion (no asparaginase activity) of circulating glutamine in live animals.

An intranasal nanoparticle vaccine elicits protective immunity against Mycobacterium tuberculosis.

Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens, thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in development. One of the primary challenges associated with mucosal vaccines has been identifying and characterizing safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity. In this study, we tested NanoSTING, a liposomal formulation of the endogenous activator of the stimulator of interferon genes (STING) pathway, cyclic guanosine adenosine monophosphate (cGAMP), as a mucosal adjuvant. We formulated a vaccine based on the H1 antigen (fusion protein of Ag85b and ESAT-6) adjuvanted with NanoSTING. Intranasal immunization of NanoSTING-H1 elicited a strong T-cell response in the lung of vaccinated animals characterized by (a) CXCR3+ KLRG1- lung resident T cells that are known to be essential for controlling bacterial infection, (b) IFNγ-secreting CD4+ T cells which is necessary for intracellular bactericidal activity, and (c) IL17-secreting CD4+ T cells that can confer protective immunity against multiple clinically relevant strains of Mtb. Upon challenge with aerosolized Mycobacterium tuberculosis Erdman strain, intranasal NanoSTING-H1 provides protection comparable to subcutaneous administration of the live attenuated Mycobacterium bovis vaccine strain Bacille-Calmette-Guérin (BCG). Our results indicate that NanoSTING adjuvanted protein vaccines can elicit a multi-factorial immune response that protects from infection by M. tuberculosis.

The efficient synthesis and purification of 2'3'- cGAMP from Escherichia coli.

Agonists of the stimulator of interferon genes (STING) pathway are being explored as potential immunotherapeutics for the treatment of cancer and as vaccine adjuvants for infectious diseases. Although chemical synthesis of 2'3' - cyclic Guanosine Monophosphate-Adenosine Monophosphate (cGAMP) is commercially feasible, the process results in low yields and utilizes organic solvents. To pursue an efficient and environmentally friendly process for the production of cGAMP, we focused on the recombinant production of cGAMP via a whole-cell biocatalysis platform utilizing the murine cyclic Guanosine monophosphate-Adenosine monophosphate synthase (mcGAS). In E. coli BL21(DE3) cells, recombinant expression of mcGAS, a DNA-dependent enzyme, led to the secretion of cGAMP to the supernatants. By evaluating the: (1) media composition, (2) supplementation of divalent cations, (3) temperature of protein expression, and (4) amino acid substitutions pertaining to DNA binding; we showed that the maximum yield of cGAMP in the supernatants was improved by 30% from 146 mg/L to 186 ± 7 mg/mL under optimized conditions. To simplify the downstream processing, we developed and validated a single-step purification process for cGAMP using anion exchange chromatography. The method does not require protein affinity chromatography and it achieved a yield of 60 ± 2 mg/L cGAMP, with <20 EU/mL (<0.3 EU/µg) of endotoxin. Unlike chemical synthesis, our method provides a route for the recombinant production of cGAMP without the need for organic solvents and supports the goal of moving toward shorter, more sustainable, and more environmentally friendly processes.

Decoding the mechanisms of chimeric antigen receptor (CAR) T cell-mediated killing of tumors: insights from granzyme and Fas inhibition.

Chimeric antigen receptor (CAR) T cell show promise in cancer treatments, but their mechanism of action is not well understood. Decoding the mechanisms used by individual T cells can help improve the efficacy of T cells while also identifying mechanisms of T cell failure leading to tumor escape. Here, we used a suite of assays including dynamic single-cell imaging of cell-cell interactions, dynamic imaging of fluorescent reporters to directly track cytotoxin activity in tumor cells, and scRNA-seq on patient infusion products to investigate the cytotoxic mechanisms used by individual CAR T cells in killing tumor cells. We show that surprisingly, overexpression of the Granzyme B (GZMB) inhibitor, protease inhibitor-9 (PI9), does not alter the cytotoxicity mediated by CD19-specific CAR T cells against either the leukemic cell line, NALM6; or the ovarian cancer cell line, SkOV3-CD19. We designed and validated reporters to directly assay T cell delivered GZMB activity in tumor cells and confirmed that while PI9 overexpression inhibits GZMB activity at the molecular level, this is not sufficient to impact the kinetics or magnitude of killing mediated by the CAR T cells. Altering cytotoxicity mediated by CAR T cells required combined inhibition of multiple pathways that are tumor cell specific: (a) B-cell lines like NALM6, Raji and Daudi were sensitive to combined GZMB and granzyme A (GZMA) inhibition; whereas (b) solid tumor targets like SkOV3-CD19 and A375-CD19 (melanoma) were sensitive to combined GZMB and Fas ligand inhibition. We realized the translational relevance of these findings by examining the scRNA-seq profiles of Tisa-cel and Axi-cel infusion products and show a significant correlation between GZMB and GZMA expression at the single-cell level in a T cell subset-dependent manner. Our findings highlight the importance of the redundancy in killing mechanisms of CAR T cells and how this redundancy is important for efficacious T cells.

Epidermal growth factor-like 7 is a novel therapeutic target in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive, noncurative, mature B-cell lymphoma, with a median overall survival of 6-7 years. This underlines a need for effective therapeutic strategies to treat MCL better. Epidermal growth factor-like 7 (EGFL7) is a protein secreted by endothelial cells shown to play a critical role in angiogenesis. Our laboratory has previously demonstrated that EGFL7 supports the growth of leukemic blasts in patients with acute myeloid leukemia (AML); however, its role in MCL has not been investigated yet. In this study, we report that EGFL7 messenger RNA (mRNA) is increased in the cells of patients with MCL compared with cells from healthy controls, and patients with high EGFL7 are associated with lower overall survival rates. Furthermore, EGFL7 is increased in the plasma of patients with MCL compared with the plasma from healthy controls. We further show that EGFL7 binds to epidermal growth factor receptor (EGFR) and activates AKT signaling pathway in MCL cells and that blocking EGFL7 in MCL in patient and cell lines decreases cell growth and increases apoptosis in vitro. Finally, anti-EGFL7 treatment inhibits tumor size and prolongs survival in a mouse model of MCL. In conclusion, our study reveals a role for EGFL7 in MCL cell proliferation and highlights EGFL7 inhibition as a promising new treatment for patients with MCL.

COVID-associated acute limb ischemia during the Delta surge and the effect of vaccines.

OBJECTIVE: Hypercoagulability is common in severe acute respiratory syndrome coronavirus 2 and has been associated with arterial thrombosis leading to acute limb ischemia (ALI). Our objective was to determine the outcomes of concurrent coronavirus disease 2019 (COVID-19) infection and ALI, particularly during the Delta variant surge and the impact of vaccination status. METHODS: A retrospective review was performed of patients treated at a single health care system between March 2020 and December 2021 for ALI and recent (<14 days) COVID-19 infection or who developed ALI during hospitalization for the same disease. Patients were grouped by year as well as by pre and post Delta variant emergence in 2021 based on the World Health Organization timeline (January to May vs June to December). Baseline demographics, imaging, interventions, and outcomes were evaluated. A control cohort of all patients with ALI requiring surgical intervention for a 2-year period prior to the pandemic was used for comparison. Primary outcomes were in-hospital mortality and amputation-free survival. Kaplan-Meier survival and Cox proportional hazards analysis were performed. RESULTS: Forty acutely ischemic limbs were identified in 36 patients with COVID-19, the majority during the Delta surge (52.8%) and after the wide availability of vaccines. The rate of COVID-19-associated ALI, although low overall, nearly doubled during the Delta surge (0.37% vs 0.20%; P = .09). Intervention (open or endovascular revascularization vs primary amputation) was performed on 31 limbs in 28 individuals, with the remaining eight treated with systemic anti-coagulation. Postoperative mortality was 48%, and overall mortality was 50%. Major amputation following revascularization was significantly higher with COVID-19 ALI (25% vs 3%; P = .006) compared with the pre-pandemic group. Thirty-day amputation-free survival was significantly lower (log-rank P < .001). COVID-19 infection (adjusted hazard ratio, 6.2; P < .001) and age (hazard ratio, 1.1; P = .006) were associated with 30-day amputation in multivariate analysis. Severity of COVID-19 infection, defined as vasopressor usage, was not associated with post-revascularization amputation. There was a higher incidence of re-thrombosis in the latter half of 2021 with the Delta surge, as reintervention for recurrent ischemia of the same limb was more common than our previous experience (21% vs 0%; P = .55). COVID-19-associated limb ischemia occurred almost exclusively in non-vaccinated patients (92%). CONCLUSIONS: ALI observed with Delta appears more resistant to standard therapy. Unvaccinated status correlated highly with ALI occurrence in the setting of COVID-19 infection. Information of limb loss as a COVID-19 complication among non-vaccinated patients may help to increase compliance.

Plasma Gut Microbe-Derived Metabolites Associated with Peripheral Artery Disease and Major Adverse Cardiac Events.

Cardiovascular diseases are associated with gut dysbiosis, but the role of microbe-derived metabolites as biomarkers or modulators of cardiovascular disease are not well understood. This is a targeted metabolomics study to investigate the association of nine microbe-derived metabolites with lower extremity peripheral artery disease (PAD), a form of atherosclerosis, and major adverse cardiac events (MACE). The study cohort consists of individuals with intermittent claudication and ankle-brachial index (ABI) < 0.9 (N = 119) and controls without clinically-apparent atherosclerosis (N = 37). The primary endpoint was MACE, a composite endpoint of myocardial infarction, coronary revascularization, stroke, transient ischemic attack, or cardiac-related death. Plasma metabolite concentrations differed significantly between the PAD and control groups. After adjustment for traditional atherosclerosis risk factors, kynurenine, hippuric acid, indole-3-propionic acid (IPA), and indole-3-aldehyde (I3A) concentrations were negatively associated with PAD, whereas indoxyl sulfate and 3-hydroxyanthranilic acid were positively associated. Hippuric acid, IPA, and I3A correlated with ABI, a surrogate for atherosclerotic disease burden. Those in the highest I3A concentration quartile had significantly improved freedom from MACE during follow-up compared to those in the lowest quartile. This study identifies specific indole- and phenyl-derived species impacted by gut microbial metabolic pathways that could represent novel microbiome-related biomarkers of PAD.

Early Growth Response Factor 1 in Aging Hematopoietic Stem Cells and Leukemia.

Aging is associated with various hematological disorders and a higher risk of myeloproliferative disorders. An aged hematopoietic system can be characterized by decreased immune function and increased myeloid cell production. Hematopoietic stem cells (HSCs) regulate the production of blood cells throughout life. The self-renewal and regenerative potential of HSCs determine the quality and quantity of the peripheral blood cells. External signals from the microenvironment under different conditions determine the fate of the HSCs to proliferate, self-renew, differentiate, or remain quiescent. HSCs respond impromptu to a vast array of extracellular signaling cascades such as cytokines, growth factors, or nutrients, which are crucial in the regulation of HSCs. Early growth response factor 1 (EGR1) is one of the key transcription factors controlling HSC proliferation and their localization in the bone marrow (BM) niche. Downregulation of Egr1 activates and recruits HSCs for their proliferation and differentiation to produce mature blood cells. Increased expression of Egr1 is implicated in immuno-aging of HSCs. However, dysregulation of Egr1 is associated with hematological malignancies such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myelogenous leukemia (CML). Here, we summarize the current understanding of the role of EGR1 in the regulation of HSC functionality and the manifestation of leukemia. We also discuss the alternative strategies to rejuvenate the aged HSCs by targeting EGR1 in different settings.

Consequences of Progressive Full-Thickness Focal Chondral Defects Involving the Medial and Lateral Femoral Condyles After Meniscectomy: A Biomechanical Study Using a Goat Model.

Background: Full-thickness chondral defects alter tibiofemoral joint homeostasis and, if left untreated, have the potential to progress to osteoarthritis. Purpose: To assess the effects of isolated and dual full-thickness chondral defect size and location on the biomechanical properties of the lateral femoral condyle (LFC) and medial femoral condyle (MFC) during dynamic knee flexion in goat knees without menisci. Methods: In 12 goat knees, we created progressively increasing full-thickness circular chondral defects (3-, 5-, and 7.5-mm diameter) in the weightbearing contact area of flexion and extension in the MFC, the LFC, or both. Each knee was fixed into a custom steel frame and attached to a motor with sensors inserted intra-articularly. For each testing condition, the knee was loaded to 100 N and underwent a dynamic range of motion between 90° of flexion and 30° of extension. The following parameters were collected: contact area, contact pressure, contact force, peak area, and peak pressure. Study Design: Controlled laboratory study. Results: The peak pressure at the defect rim of the MFC at full extension increased by 51.51% from no defect (1.887 MPa) to a 7.5-mm defect (2.859 MPa) (P < .001), and the peak pressure at the defect rim of the LFC at full extension increased by 139.14% from no defect (1.704 MPa) to a 7.5-mm defect (4.075 MPa) (P < .001). The peak pressures for LFC defects at all 3 diameters were significantly greater when compared with dual defects consisting of increasing LFC defect diameter and constant MFC defect diameter (P < .001 for all). Conclusion: Extremely large increases in peak pressure were seen at the rim of articular cartilage defects when evaluated under dynamic loading conditions. Isolated LFC defects experienced a greater increase in defect rim stress concentrations when compared with isolated MFC defects for equivalent increases in defect size. Defect size played a significant role independent of location for peak pressures on the MFC and LFC. Clinical Relevance: Significant rim-loading effects increase with defect size under dynamic loading and may result in increasingly rapid progression of articular cartilage lesions. Within the context of this goat model, findings suggest that lateral compartment chondral lesions are more likely to progress than medial compartment lesions of equivalent size.

Modulating endothelial cells with EGFL7 to diminish aGVHD after allogeneic bone marrow transplantation in mice.

Acute graft-versus-host disease (aGVHD) is the second most common cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT), underscoring the need for novel therapies. Based on previous work that endothelial cell dysfunction is present in aGVHD and that epidermal growth factor-like domain 7 (EGFL7) plays a significant role in decreasing inflammation by repressing endothelial cell activation and T-cell migration, we hypothesized that increasing EGFL7 levels after allo-HSCT will diminish the severity of aGVHD. Here, we show that treatment with recombinant EGFL7 (rEGFL7) in 2 different murine models of aGVHD decreases aGVHD severity and improves survival in recipient mice after allogeneic transplantation with respect to controls without affecting graft-versus-leukemia effect. Furthermore, we showed that rEGFL7 treatment results in higher thymocytes, T, B, and dendritic cell counts in recipient mice after allo-HSCT. This study constitutes a proof of concept of the ability of rEGFL7 therapy to reduce GHVD severity and mortality after allo-HSCT.

Three cases of acute venous thromboembolism in females after vaccination for coronavirus disease 2019.

Since December 2020, four vaccines for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) have been developed, and three have been approved for immediate use in the United States. Two are mRNA vaccines, and one uses a viral vector mechanism. Thrombotic complications have been reported after vaccine administration, which were primarily cerebral sinus thromboses after administration of the viral vector vaccines. To the best of our knowledge, we are the first to report venous thrombotic complications within days of administration of the mRNA-1273 (Moderna) vaccine. We present a series of three women who developed venous thromboembolism after RNA-1273 vaccination at a single healthcare system.

Structural Remodeling of the Extracellular Matrix in Arteriogenesis: A Review.

Lower extremity arterial occlusive disease (AOD) results in significant morbidity and mortality for the population, with up to 10% of patients ultimately requiring amputation. An alternative method for non-surgical revascularization which is yet to be fully understood is the optimization of the body's own natural collateral arterial network in a process known as arteriogenesis. Under conditions of conductance vessel stenosis or occlusion resulting in increased flow, shear forces, and pressure gradients within collaterals, positive remodeling occurs to increase the diameter and capacity of these vessels. The creation of a distal arteriovenous fistula (AVF) will drive increased arteriogenesis as compared to collateral formation with the occlusion of a conductance vessel alone by further increasing flow through these arterioles, demonstrating the capacity for arteriogenesis to form larger, more efficient collaterals beyond what is spontaneously achieved after arterial occlusion. Arteries rely on an extracellular matrix (ECM) composed of elastic fibers and collagens that provide stability under hemodynamic stress, and ECM remodeling is necessary to allow for increased diameter and flow conductance in mature arterial structures. When positive remodeling occurs, digestion of lamella and the internal elastic lamina (IEL) by matrix metalloproteinases (MMPs) and other elastases results in the rearrangement and thinning of elastic structures and may be replaced with disordered elastin synthesis without recovery of elastic function. This results in transmission of wall strain to collagen and potential for aneurysmal degeneration along collateral networks, as is seen in the pancreaticoduodenal artery (PDA) after celiac occlusion and inferior mesenteric artery (IMA) with concurrent celiac and superior mesenteric artery (SMA) occlusions. Further understanding into the development of collaterals is required to both better understand aneurysmal degeneration and optimize collateral formation in AOD.

Curcumin restores the engraftment capacity of aged hematopoietic stem cells and also reduces PD-1 expression on cytotoxic T cells.

Aging affects the functionality of hematopoietic stem cells (HSCs), and therefore, aged individuals are not preferred as donors in HSC transplantation. Such elimination leads to the restriction of donor cohort. Several efforts are being done to rejuvenate aged HSCs. Here, we show that treatment of aged mice with curcumin rejuvenates their HSCs by restoring the expression of autophagy-inducing messenger RNAs in them, and improves their engraftment capacity. Importantly, we show that curcumin is effective in rejuvenation of HSCs when administered via both, intraperitoneal as well as oral routes. Aging also affects the immune system. While elderly individuals are not immuno-deficient, they do not respond optimally to immunizations, and hence, a strategy needs to be developed to make them immunologically responsive. Programmed cell death 1 (PD-1), one of the inhibitory coreceptors, plays an important role in the regulation of autoimmunity, infectious immunity, and cancer immunity. Its expression on T cells is indicative of their exhaustion. Here, we show that curcumin reduces the frequency of PD1+ cytotoxic T cells in the spleens of aged mice. Curcumin has a proven safety profile, and hence, can be used to treat aged donors to boost the functionality of their HSCs and also to improve the immunological profile of aged individuals. These data could have implications in various other regenerative medicine protocols as well.

Simultaneous Upregulation of Elastolytic and Elastogenic Factors Are Necessary for Regulated Collateral Diameter Expansion.

Background: During arteriogenesis, outward remodeling of the arterial wall expands luminal diameter to produce increased conductance in developing collaterals. We have previously shown that diameter expansion without loss of internal elastic lamina (IEL) integrity requires both degradation of elastic fibers and LOX-mediated repair. The aim of this study was to investigate the expression of genes involved in remodeling of the extracellular matrix (ECM) using a model of arteriogenesis. Methods: Sprague-Dawley rats underwent femoral artery ligation with distal arteriovenous fistula (FAL + AVF) placement. Profunda femoral arteries (PFA) were harvested for analysis at various time points. Serum desmosine, an amino acid found exclusively in elastin, was evaluated with enzyme-linked immunosorbent assay (ELISA) as a marker of tissue elastolysis. Tissue mRNA isolated from FAL + AVF exposed PFAs was compared to the contralateral sham-operated using qPCR. HCAECs were cultured under low shear stress (8 dyn·s/cm 2) for 24 h and then exposed to high shear stress (40 dyn·s/cm 2) for 2-6 h. Primers used included FBN-1, FBN-2, Timp-2, LOX-1, Trop-E, Cath-K, Cath-S, MMP-2, MMP-9, FBLN-4, and FBLN-5 and were normalized to GAPDH. mRNA fold changes were quantified using the 2-ΔΔCq method. Comparisons between time points were made with non-parametric ANOVA analysis with Bonferroni adjustment. Results: PFAs showed IEL reorganization during arteriogenesis. Serum desmosine levels are significantly elevated at 2 days and one week, with a return to baseline thereafter (p < 0.01). Expression of ECM structural proteins (FBN-1, FBN-2, FBLN-4, FBLN-5, Tropoelastin, TIMP-2, LOX-1) and elastolytic proteins (MMP-2, MMP-9, Cathepsin S, Cathepsin K) exhibited an early peak (p < 0.05) relative to sham PFAs. After two weeks, expression returned to baseline. HCAECs demonstrated upregulation of FBN-2, FBLN-5, LOX-1 and Trop-E at 4 h of high shear stress, as well as elastolytic protein MMP-2. Conclusions: Elastin degradation begins early in arteriogenesis and is mediated by local upregulation of elastolytic genes. Elastolysis appears to be simultaneously balanced by production of elastic fiber components which may facilitate stabilization of the IEL. Endothelial cells are central to initiation of arteriogenesis and begin ECM remodeling in response to altered shear stress.

Surgical Resection of a Symptomatic Superior Vena Cava Lipoma: A Case Report and Literature Review.

BACKGROUND: Lipomas are the most common form of benign soft tissue neoplasms and most frequently occur in the subcutaneous tissue. Rarely does a lipoma primarily arise from the arteries or veins. The most common location for an intravascular lipoma is the inferior vena cava, and rarely lipomas originate in the superior vena cava (SVC). Large lipomas of the SVC may be associated with central venous occlusive symptoms. There are only 7 cases of SVC lipomas reported in the literature. Here, we present only the second case of a large symptomatic lipoma located in the SVC, right internal jugular vein, and innominate veins. METHODS: We present a case of a 5-cm lipoma located in the SVC, discovered incidentally and surgically resected via median sternotomy. RESULTS: The patient underwent a successful open surgical resection of a symptomatic lipoma located in his SVC. CONCLUSIONS: Lipomas of the SVC are exceptionally rare, with only 7 cases described in the literature. This case demonstrates that lipomas can be safely excised from the SVC leading to resolution of central venous occlusive symptoms. A comprehensive literature review reveals that surgical resection is generally without complication, leads to resolution of symptoms, and does not require long-term follow-up.

Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing.

The ability to accurately measure mutations is critical for basic research and identifying potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and do not fully represent other species such as humans. Next-generation sequencing (NGS) is a conceptually attractive alternative for detecting mutations in the DNA of any organism; however, the limit of resolution for standard NGS is poor. Technical error rates (∼1 × 10-3) of NGS obscure the true abundance of somatic mutations, which can exist at per-nucleotide frequencies ≤1 × 10-7 Using duplex sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by three carcinogens in five tissues of two strains of mice within 31 d following exposure. We observed a strong correlation between mutation induction measured by duplex sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified a primordial marker of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex sequencing can be broadly applied to basic mutational research, regulatory safety testing, and emerging clinical applications.

Methylation-dependent antioxidant-redox imbalance regulates hypertensive kidney injury in aging.

The prevalence of hypertension increases with age, and oxidative stress is a major contributing factor to the pathogenesis of hypertension-induced kidney damage in aging. The nicotinamide adenine dinucleotide phosphate (NADPH) family is one of the major sources of reactive oxygen species (ROS) generation, and several NADPH oxidase isoforms are highly expressed in the kidney. Although epigenetic protein modification plays a role in organ injury, the methylation of the oxidant-antioxidant defense system and their role in hypertension-induced kidney damage in aging remains underexplored. The present study investigated the role of NADPH oxidase 4, superoxide dismutases (SODs), catalase, and NOS in Ang-II induced kidney damage in aging. Wild type (WT, C57BL/6J) mice aged 12-14 and 75-78 weeks were used and treated with or without Ang-II (1000 ng/kg/min) for 4 weeks with control mice receiving saline. Aged mice with or without Ang-II exhibited higher mean BP, lower renal blood flow, and decreased renal vascular density compared to young mice. While superoxide, 4-HNE, p22phox, Nox4, iNOS were increased in the aged kidney, the expression of eNOS, MnSOD, CuSOD, catalase, Sirt1, and -3 as well as the ratio of GSH/GSSG, and activities of SODs and catalase were decreased compared to young control mice. The changes further deteriorated with Ang-II treatment. In Ang-II treated aged mice, the expressions of DNMTs were increased and associated with increased methylation of SODs, Sirt1, and Nox4. We conclude that hypermethylation of antioxidant enzymes in the aged kidney during hypertension worsens redox imbalance leading to kidney damage.

Comparable Patency of Open and Hybrid Treatment of Venous Anastomotic Lesions in Thrombosed Haemodialysis Grafts.

OBJECTIVE: Arteriovenous graft (AVG) failures are typically associated with venous anastomotic (VA) stenosis. Current evidence regarding AVG thrombosis management compares surgical with purely endovascular techniques; few studies have investigated the "hybrid" intervention that combines surgical balloon thrombectomy and endovascular angioplasty and/or stenting to address VA obstruction. This study aimed to describe outcomes after hybrid intervention compared with open revision (patch venoplasty or jump bypass) of the VA in thrombosed AVGs. METHODS: Retrospective cohort study. Consecutive patients with a thrombosed AVG who underwent thrombectomy between January 2014 and July 2018 were divided into open and hybrid groups based on VA intervention; patients who underwent purely endovascular thrombectomy were excluded. Patient demographics, previous access history, central vein patency, AVG anatomy, type of intervention, and follow up data were recorded. Kaplan-Meier curves were used to analyse time from thrombectomy to first re-intervention (primary patency) and time to abandonment (secondary patency). Cox regression analysis was performed to evaluate predictors of failure. RESULTS: This study included 97 patients (54 females) with 39 forearm, 47 upper arm, and 11 lower extremity AVGs. There were 34 open revisions (25 patches, nine jump bypasses) and 63 hybrid interventions, which included balloon angioplasty ± adjunctive procedures (15 stents, five cutting balloons). Technique selection was based on physician preference. Primary patency for the open and hybrid groups was 27.8% and 34.2%, respectively, at six months and 17.5% and 12.9%, respectively, at 12 months (p = .71). Secondary patency was 45.1% and 38.5% for open and hybrid treatment, respectively, at 12 months (p = .87). An existing VA stent was predictive of graft abandonment (hazard ratio 4.4, 95% confidence interval 1.2-16.0; p = .024). Open vs. hybrid intervention was not predictive of failure or abandonment. CONCLUSION: Hybrid interventions for thrombosed AVGs are not associated with worse patency at six and 12 months compared with open revision.