Skeletal Muscle, Skin, and Bone as Three Major Nitrate Reservoirs in Mammals: Chemiluminescence and 15N-Tracer Studies in Yorkshire Pigs.

In mammals, nitric oxide (NO) is generated either by the nitric oxide synthase (NOS) enzymes from arginine or by the reduction of nitrate to nitrite by tissue xanthine oxidoreductase (XOR) and the microbiome and further reducing nitrite to NO by XOR or several heme proteins. Previously, we reported that skeletal muscle acts as a large nitrate reservoir in mammals, and this nitrate reservoir is systemically, as well as locally, used to generate nitrite and NO. Here, we report identifying two additional nitrate storage organs-bone and skin. We used bolus of ingested 15N-labeled nitrate to trace its short-term fluxes and distribution among organs. At baseline conditions, the nitrate concentration in femur bone samples was 96 ± 63 nmol/g, scalp skin 56 ± 22 nmol/g, with gluteus muscle at 57 ± 39 nmol/g. In comparison, plasma and liver contained 34 ± 19 nmol/g and 15 ± 5 nmol/g of nitrate, respectively. Three hours after 15N-nitrate ingestion, its concentration significantly increased in all organs, exceeding the baseline levels in plasma, skin, bone, skeletal muscle, and in liver 5-, 2.4-, 2.4-, 2.1-, and 2-fold, respectively. As expected, nitrate reduction into nitrite was highest in liver but also substantial in skin and skeletal muscle, followed by the distribution of 15N-labeled nitrite. We believe that these results underline the major roles played by skeletal muscle, skin, and bone, the three largest organs in mammals, in maintaining NO homeostasis, especially via the nitrate-nitrite-NO pathway.

Dietary Nitrate Metabolism in Porcine Ocular Tissues Determined Using 15N-Labeled Sodium Nitrate Supplementation.

Nitrate (NO3-) obtained from the diet is converted to nitrite (NO2-) and subsequently to nitric oxide (NO) within the body. Previously, we showed that porcine eye components contain substantial amounts of nitrate and nitrite that are similar to those in blood. Notably, cornea and sclera exhibited the capability to reduce nitrate to nitrite. To gain deeper insights into nitrate metabolism in porcine eyes, our current study involved feeding pigs either NaCl or Na15NO3 and assessing the levels of total and 15N-labeled NO3-/NO2- in various ocular tissues. Three hours after Na15NO3 ingestion, a marked increase in 15NO3- and 15NO2- was observed in all parts of the eye; in particular, the aqueous and vitreous humor showed a high 15NO3- enrichment (77.5 and 74.5%, respectively), similar to that of plasma (77.1%) and showed an even higher 15NO2- enrichment (39.9 and 35.3%, respectively) than that of plasma (19.8%). The total amounts of NO3- and NO2- exhibited patterns consistent with those observed in 15N analysis. Next, to investigate whether nitrate or nitrite accumulate proportionally after multiple nitrate treatments, we measured nitrate and nitrite contents after supplementing pigs with Na15NO3 for five consecutive days. In both 15N-labeled and total nitrate and nitrite analysis, we did not observe further accumulation of these ions after multiple treatments, compared to a single treatment. These findings suggest that dietary nitrate supplementation exerts a significant influence on nitrate and nitrite levels and potentially NO levels in the eye and opens up the possibility for the therapeutic use of dietary nitrate/nitrite to enhance or restore NO levels in ocular tissues.

Potential roles of nitrate and nitrite in nitric oxide metabolism in the eye.

Nitric oxide (NO) signaling has been studied in the eye, including in the pathophysiology of some eye diseases. While NO production by nitric oxide synthase (NOS) enzymes in the eye has been characterized, the more recently described pathways of NO generation by nitrate (NO3-) and nitrite (NO2-) ions reduction has received much less attention. To elucidate the potential roles of these pathways, we analyzed nitrate and nitrite levels in components of the eye and lacrimal glands, primarily in porcine samples. Nitrate and nitrite levels were higher in cornea than in other eye parts, while lens contained the least amounts. Lacrimal glands exhibited much higher levels of both ions compared to other organs, such as liver and skeletal muscle, and even to salivary glands which are known to concentrate these ions. Western blotting showed expression of sialin, a known nitrate transporter, in the lacrimal glands and other eye components, and also xanthine oxidoreductase, a nitrate and nitrite reductase, in cornea and sclera. Cornea and sclera homogenates possessed a measurable amount of nitrate reduction activity. These results suggest that nitrate ions are concentrated in the lacrimal glands by sialin and can be secreted into eye components via tears and then reduced to nitrite and NO, thereby being an important source of NO in the eye.

CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis.

Intake of hemoglobin by the hemoglobin-haptoglobin receptor CD163 leads to a distinct alternative non-foam cell antiinflammatory macrophage phenotype that was previously considered atheroprotective. Here, we reveal an unexpected but important pathogenic role for these macrophages in atherosclerosis. Using human atherosclerotic samples, cultured cells, and a mouse model of advanced atherosclerosis, we investigated the role of intraplaque hemorrhage on macrophage function with respect to angiogenesis, vascular permeability, inflammation, and plaque progression. In human atherosclerotic lesions, CD163+ macrophages were associated with plaque progression, microvascularity, and a high level of HIF1α and VEGF-A expression. We observed irregular vascular endothelial cadherin in intraplaque microvessels surrounded by CD163+ macrophages. Within these cells, activation of HIF1α via inhibition of prolyl hydroxylases promoted VEGF-mediated increases in intraplaque angiogenesis, vascular permeability, and inflammatory cell recruitment. CD163+ macrophages increased intraplaque endothelial VCAM expression and plaque inflammation. Subjects with homozygous minor alleles of the SNP rs7136716 had elevated microvessel density, increased expression of CD163 in ruptured coronary plaques, and a higher risk of myocardial infarction and coronary heart disease in population cohorts. Thus, our findings highlight a nonlipid-driven mechanism by which alternative macrophages promote plaque angiogenesis, leakiness, inflammation, and progression via the CD163/HIF1α/VEGF-A pathway.

Repair of Tympanic Membrane Perforations with Customized Bioprinted Ear Grafts Using Chinchilla Models.

The goal of this work is to develop an innovative method that combines bioprinting and endoscopic imaging to repair tympanic membrane perforations (TMPs). TMPs are a serious health issue because they can lead to both conductive hearing loss and repeated otitis media. TMPs occur in 3-5% of cases after ear tube placement, as well as in cases of acute otitis media (the second most common infection in pediatrics), chronic otitis media with or without cholesteatoma, or as a result of barotrauma to the ear. About 55,000 tympanoplasties, the surgery performed to reconstruct TMPs, are performed every year, and the commonly used cartilage grafting technique has a success rate between 43% and 100%. This wide variability in successful tympanoplasty indicates that the current approach relies heavily on the skill of the surgeon to carve the shield graft into the shape of the TMP, which can be extremely difficult because of the perforation's irregular shape. To this end, we hypothesized that patient specific acellular grafts can be bioprinted to repair TMPs. In vitro data demonstrated that our approach resulted in excellent wound healing responses (e.g., cell invasion and proliferations) using our bioprinted gelatin methacrylate constructs. Based on these results, we then bioprinted customized acellular grafts to treat TMP based on endoscopic imaging of the perforation and demonstrated improved TMP healing in a chinchilla study. These ear graft techniques could transform clinical practice by eliminating the need for hand-carved grafts. To our knowledge, this is the first proof of concept of using bioprinting and endoscopic imaging to fabricate customized grafts to treat tissue perforations. This technology could be transferred to other medical pathologies and be used to rapidly scan internal organs such as intestines for microperforations, brain covering (Dura mater) for determination of sites of potential cerebrospinal fluid leaks, and vascular systems to determine arterial wall damage before aneurysm rupture in strokes.

"On-command" dissolvable tympanostomy tube in the chinchilla model: A proof of concept.

OBJECTIVES: To prove the concept that a dissolvable "on-command" tympanostomy tube placed into the tympanic membrane of a chinchilla can dissolve when a benign solution is applied and result in a well healed tympanic membrane without histologic evidence of injury. STUDY DESIGN: Prospective Randomized Single-Subject Controlled Trial. METHODS: Prototype tympanostomy tubes were fabricated from poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylate-co- methyl methacrylate) (PBM). "In vitro" dissolution studies were performed with applications of the benign chemical, hydrogen peroxide (HP). PBM tubes were placed into ten chinchilla tympanic membranes matched with standard plastic tubes placed into the contralateral side. All 20 tubes were exposed to HP for 21 days with serial endoscopic examinations. In vitro PBM tubes were weighed before and after interventions and compared to control tubes. In vivo photo documentation was used to show progression of dissolution and histologic slides were obtained to show the effect of the PBM on surrounding tissues. RESULTS: Compared to control tubes, all those exposed to hydrogen peroxide had a statistically significant reduction in weight (p < 0.01). After placement into the tympanic membrane of chinchillas, all PBM tubes dissolved within 21 days of hydrogen peroxide treatment leaving behind histologically normal, intact tympanic membranes. CONCLUSION: Our PBM tubes dissolve "on-command" in a chinchilla model when exposed to treatment with a benign chemical. Dissolvable "on-command" tympanostomy tubes may reduce significant complications related to pediatric tympanostomy tube use.

Apigenin inhibits COX-2, PGE2, and EP1 and also initiates terminal differentiation in the epidermis of tumor bearing mice.

Non-melanoma skin cancer (NMSC) is the most prevalent cancer in the United States. NMSC overexpresses cyclooxygenase-2 (COX-2). COX-2 synthesizes prostaglandins such as PGE2 which promote proliferation and tumorigenesis by engaging G-protein-coupled prostaglandin E receptors (EP). Apigenin is a bioflavonoid that blocks mouse skin tumorigenesis induced by the chemical carcinogens, 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the effect of apigenin on the COX-2 pathway has not been examined in the DMBA/TPA skin tumor model. In the present study, apigenin decreased tumor multiplicity and incidence in DMBA/TPA-treated SKH-1 mice. Analysis of the non-tumor epidermis revealed that apigenin reduced COX-2, PGE2, EP1, and EP2 synthesis and also increased terminal differentiation. In contrast, apigenin did not inhibit the COX-2 pathway or promote terminal differentiation in the tumors. Since fewer tumors developed in apigenin-treated animals which contained reduced epidermal COX-2 levels, our data suggest that apigenin may avert skin tumor development by blocking COX-2.

Expression of Bacteroides fragilis hemolysins in vivo and role of HlyBA in an intra-abdominal infection model.

Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infections. However, there is a paucity of information regarding the genetic and molecular aspects of gene expression of its virulence factors during extra-intestinal infections. A potential virulence factor that has received little attention is the ability of B. fragilis to produce hemolysins. In this study, an implanted perforated table tennis "ping-pong" ball was used as an intra-abdominal artificial abscess model in the rat. This procedure provided sufficient infected exudate for gene expression studies in vivo. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify the relative expression of hlyA, hlyB, hlyC, hlyD, hlyE, hlyF, hlyG, and hlyIII mRNAs. The hlyA mRNA was induced approximately sixfold after 4 days postinfection compared with the mRNA levels in the inoculum culture prior to infection. The hlyB mRNA increased approximately sixfold after 4 days and 12-fold after 8 days postinfection. Expression of hlyC mRNA increased sixfold after 1 day, 45-fold after 4 days, and 16-fold after 8 days postinfection, respectively. The hlyD and hlyE mRNAs were induced approximately 40-fold and 30-fold, respectively, after 4-days postinfection. The hlyF expression increased approximately threefold after 4-days postinfection. hlyG was induced approximately fivefold after 4 and 8 days postinfection. The hlyIII mRNA levels had a steady increase of approximately four-, eight-, and 12-fold following 1, 4, and 8 days postinfection, respectively. These findings suggest that B. fragilis hemolysins are induced and differentially regulated in vivo. Both parent and hlyBA mutant strains reached levels of approximately 3-8 × 10(9) cfu/mL after 1 day postinfection. However, the hlyBA mutant strain lost 2 logs in viable cell counts compared with the parent strain after 8 days postinfection. This is the first study showing HlyBA is a virulence factor which plays a role in B. fragilis survival in an intra-abdominal abscess model.

Arachidonoyl ethanolamide (AEA)-induced apoptosis is mediated by J-series prostaglandins and is enhanced by fatty acid amide hydrolase (FAAH) blockade.

The endocannabinoid arachidonoyl ethanolamide (AEA) is a potent inducer of tumor cell apoptosis however its mechanism of cytotoxicity is unclear. A previous report from our laboratory showed that AEA induced cell death in a cyclooxygenase-2 (COX-2)-dependent manner and in this report our data indicate that AEA-induced apoptosis is mediated by COX-2 metabolic products of the J-series. In experiments conducted with JWF2 keratinocytes which over-express COX-2, AEA caused a concentration-regulated increase in J-series prostaglandin production and apoptosis. Similarly, cell treatment with exogenously added J-series prostaglandins (15-deoxy, Δ(12,14) PGJ(2) and PGJ(2)) induced apoptosis. AEA-induced apoptosis was inhibited by the antioxidant, N-acetyl cysteine, indicating that reactive oxygen species generation was required for apoptosis. Using antagonists of cannabinoid receptor 1, cannabinoid receptor 2, or transient receptor potential cation channel, subfamily V, member 1, it was observed that cannabinoid receptor inhibition did not block AEA-mediated cell death. In contrast, an inhibitor of fatty acid amide hydrolase (FAAH) potentiated AEA-induced J-series PG synthesis and apoptosis. These results suggest that the metabolism of AEA to J-series PGs regulates the induction of apoptosis in cells with elevated COX-2 levels. Our data further indicate that the proapoptotic activity of AEA can be enhanced by combining it with an inhibitor of FAAH. As such, AEA may be an effective agent to eliminate tumor cells that over-express COX-2.

Surfactant protein A and B genetic variants predispose to idiopathic pulmonary fibrosis.

Derangement in pulmonary surfactant or its components and alveolar collapse are common findings in idiopathic pulmonary fibrosis (IPF). Surfactant proteins play important roles in innate host defense and normal function of the lung. We examined associations between IPF and genetic polymorphic variants of surfactant proteins, SP-A1, SP-A2, SP-B, SP-C, and SP-D. One SP-A1 (6A(4)) allele and single nucleotide polymorphisms (SNPs) that characterize the 6A(4) allele, and one SP-B (B1580_C) were found with higher frequency ( P

Mucosally-derived HPV-40 can infect both human genital foreskin and cutaneous hand skin tissues grafted into athymic mice.

HPV-40 is a rare HPV type that has been detected only in genital mucosal tissues. This HPV type is very closely related to HPV-7, which has a predominantly cutaneous tissue tropism. We have shown, previously, that an isolate of HPV-40 (described here as HPV-40(Hershey) or HPV-40(H)) productively infected genital tissues. In this study, HPV-40(H) was tested for productive infection of cutaneous tissue. Fetal hand skin fragments were incubated with infectious HPV-40(H) and implanted subrenally into athymic mice. After 120 days, xenografts showed morphological changes consistent with HPV-40(H) infection and were HPV-40 DNA in situ positive and capsid antigen positive. The results demonstrated that hand skin can support HPV-40(H) infection thereby indicating that this viral type has the capacity to infect both genital mucosal and cutaneous tissues.