Supraphysiological testosterone enanthate administration prevents bone loss and augments bone strength in gonadectomized male and female rats

High-dose testosterone enanthate (TE) may prevent hypogonadism-induced osteopenia. For this study, 3-mo-old male and female Fisher SAS rats underwent sham surgery, gonadectomy (GX), or GX plus 28 days TE administration (7.0 mg/wk). GX reduced serum sex hormones (i.e., testosterone, dihydrotestosterone, and estradiol) (P < 0.05) in both sexes and bone concentrations of testosterone (males only), and estradiol (females only). GX also elevated urine deoxypyridinoline/creatinine in both sexes and serum osteocalcin (females only), findings that are consistent with high-turnover osteopenia. GX reduced cancellous bone volume (CBV) and increased osteoid surfaces in tibia of both sexes. GX males also experienced reduced trabecular number and width and increased trabecular separation, whereas GX females experienced increased osteoblast and osteoid surfaces. Bone biomechanical characteristics remained unaffected by GX, except that femoral stiffness was reduced in females. In contrast, TE administration to GX rats elevated serum and bone androgens to supraphysiological concentrations in both sexes but altered neither serum nor bone estradiol in males. Additionally, TE did not prevent GX-induced reductions in serum or bone estradiol in females. TE also reduced markers of high-turnover osteopenia in both sexes. In males, TE prevented GX-induced changes in trabecular number and separation, CBV, and osteoid surfaces while diminishing osteoblast and osteoclast surfaces; however, these changes were not fully prevented in females. In both sexes, TE increased femoral length and femoral maximal strength to above that of Sham and GX animals while preventing the loss of femoral stiffness in females. In conclusion, TE administration appears protective of cancellous bone in male rats and augments cortical bone strength in both sexes.     

Lymph flow in the thoracic duct of conscious dogs during lymphatic pump treatment, exercise, and expansion of extracellular fluid volume

BACKGROUND: This investigation examined interactions between expansion of the extracellular fluid volume (ECE), osteopathic lymphatic pump treatment (LPT), and exercise on lymph flow in the thoracic duct of eight instrumented, conscious dogs. METHODS AND RESULTS: After recovery from surgery, LPT was performed for 8 min before and after ECE with normal saline, i.v., 4.4+/-0.3% of body weight. Baseline lymph flow was 1.7+/-0.5 mL/min. LPT rapidly increased lymph flow to 5.0+/-1.1 mL/min at 1 min, and lymph flow remained above baseline for 4 min (p<0.05). LPT produced a net increase in lymph flow of 15.4+/-1.1 mL. Following ECE, baseline lymph flow was 4.8+/-0.6 mL/min (p<0.05). LPT increased lymph flow to 9.9+/-1.1 mL/min at 1 min (p<0.05), and lymph flow remained above baseline for 4 min (p<0.05); all flow values after ECE were greater than corresponding values before ECE. However, the net increase in lymph flow produced by 8 min of LPT (18.3+/-3.8 mL) was not significantly greater than that observed before ECE. Moderate treadmill exercise increased lymph flow for 4 min before ECE and for 6 min after ECE. All lymph flows during exercise were greater after ECE than before ECE. The net increase in lymph flow produced by 8 min of exercise was 24.9+/-5.5 mL before ECE and 39.6+/-5.1 mL after ECE (p<0.05). CONCLUSIONS: Expansion of the extracellular fluid volume produced large increases in thoracic duct lymph flow, that were further augmented by lymphatic pump treatment and by moderate treadmill exercise.     

Design,synthesis, and biological evaluation of inhibitors of the NADPH oxidase,Nox4

NADPH oxidases (Nox enzymes) are critical mediators of both physiologic and pathophysiologic processes. Nox enzymes catalyze NADPH-dependent generation of reactive oxygen species (ROS), including superoxide and hydrogen peroxide. Until recently, Nox4 was proposed to be involved exclusively in normal physiologic functions. Compelling evidence, however, suggests that Nox4 plays a critical role in fibrosis, as well as a host of pathologies and diseases. These considerations led to a search for novel, small molecule inhibitors of this important enzyme. Ultimately, a series of novel tertiary sulfonylureas (23–25) was designed using pharmacophore modeling, synthesized, and evaluated for inhibition of Nox4-dependent signaling.     

Controlling herpes simplex virus-induced ocular inflammatory lesions with the lipid-derived mediator resolvin E1

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye caused by HSV-1 infection and a common cause of blindness in humans. The inflammatory lesions are primarily perpetuated by neutrophils with the active participation of CD4(+) T cells. Therefore, targeting these immune cell types represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin E1 (RvE1), an endogenous lipid mediator, was shown to promote resolution in several inflammatory disease models. In the current report, we determined whether RvE1 administration begun at different times after ocular infection of mice with HSV could influence the severity of SK lesions. Treatment with RvE1 significantly reduced the extent of angiogenesis and SK lesions that occurred. RvE1-treated mice had fewer numbers of inflammatory cells that included Th1 and Th17 cells as well as neutrophils in the cornea. The mechanisms by which RvE1 acts appear to be multiple. These included reducing the influx of neutrophils and pathogenic CD4(+) T cells, increasing production of the anti-inflammatory cytokine IL-10, and inhibitory effects on the production of proinflammatory mediators and molecules, such as IL-6, IFN-γ, IL-17, KC, VEGF-A, MMP-2, and MMP-9, that are involved in corneal neovascularization and SK pathogenesis. These findings are, to our knowledge, the first to show that RvE1 treatment could represent a novel approach to control lesion severity in a virally induced immunopathological disease.     

Ocular neovascularization caused by herpes simplex virus type 1 infection results from breakdown of binding between vascular endothelial growth factor A and its soluble receptor

The normal cornea is transparent, which is essential for normal vision, and although the angiogenic factor vascular endothelial growth factor A (VEGF-A) is present in the cornea, its angiogenic activity is impeded by being bound to a soluble form of the VEGF receptor-1 (sVR-1). This report investigates the effect on the balance between VEGF-A and sVR-1 that occurs after ocular infection with HSV, which causes prominent neovascularization, an essential step in the pathogenesis of the vision-impairing lesion, stromal keratitis. We demonstrate that HSV-1 infection causes increased production of VEGF-A but reduces sVR-1 levels, resulting in an imbalance of VEGF-A and sVR-1 levels in ocular tissues. Moreover, the sVR-1 protein made was degraded by the metalloproteinase (MMP) enzymes MMP-2, -7, and -9 produced by infiltrating inflammatory cells that were principally neutrophils. Inhibition of neutrophils, inhibition of sVR-1 breakdown with the MMP inhibitor marimastat, and the provision of exogenous recombinant sVR-1 protein all resulted in reduced angiogenesis. Our results make the novel observation that ocular neovascularization resulting from HSV infection involves a change in the balance between VEGF-A and its soluble inhibitory receptor. Future therapies aimed to increase the production and activity of sVR-1 protein could benefit the management of stromal keratitis, an important cause of human blindness.     

Effect of novel synthetic evodiamine analogue on sexual behavior in male rats

Currently phosphodiestrase5 (PDE5) inhibitors are the first-line treatment for erectile dysfunction. Drugs such as sildenafil and tadalafil are available as PDE5 inhibitors which are potent and reversible but lack selectivity with side effects such as headache, facial flushing, dyspepsia, and visual disturbances. We herein report for the first time novel condensed thienopyrimidines as evodiamine analogue and their effect on sexual behavior in male rats hitherto unreported. Novel synthetic evodiamine significantly showed improvement in male rat copulatory behavior. The test compound MKAC9 could be of promising importance in the treatment of sexual disorders like desire disorder or erectile dysfunction.

Unique sphingomyelin patches are targets of a beta-cell-specific antibody

To devise successful imaging and therapeutic strategies, the identification of β-cell surface markers is one of the challenges in diabetes research that has to be resolved. We previously showed that IC2, a rat monoclonal IgM antibody, can be used for ex vivo determination of β-cell mass by imaging. Further progress toward the development of an antibody-based imaging agent was hampered by the lack of knowledge regarding the nature and composition of the IC2 antigen. Here, we show a series of systematic experiments involving classical lipid extraction and chromatography techniques combined with immunochemistry, which led to the identification of sphingomyelin as the target antigen assembled in the form of patches on the β-cell surface. Our findings were verified by modulating SM by enzymatic cleavage, downregulation, upregulation, and perturbation of membrane SM and observation of corresponding changes in IC2 binding. Cholesterol participates in stabilization of these patches, as its removal results in loss of IC2 binding. We believe that these findings have implications for identifying future ligands for the proposed antigen for imaging purposes as well as for potential therapy, as sphingomyelin has been shown to play a role in the apoptotic cascade in pancreatic β cells.     

An anti-inflammatory role of VEGFR2/Src kinase inhibitor in herpes simplex virus 1-induced immunopathology

Corneal neovascularization represents a key step in the blinding inflammatory stromal keratitis (SK) lesion caused by ocular infection with herpes simplex virus (HSV). In this report, we describe a novel approach for limiting the angiogenesis caused by HSV infection of the mouse eye. We show that topical or systemic administration of the Src kinase inhibitor (TG100572) that inhibits downstream molecules involved in the vascular endothelial growth factor (VEGF) signaling pathway resulted in markedly diminished levels of HSV-induced angiogenesis and significantly reduced the severity of SK lesions. Multiple mechanisms were involved in the inhibitory effects. These included blockade of IL-8/CXCL1 involved in inflammatory cells recruitment that are a source of VEGF, diminished cellular infiltration in the cornea, and reduced proliferation and migration of CD4(+) T cells into the corneas. As multiple angiogenic factors (VEGF and basic fibroblast growth factor [bFGF]) play a role in promoting angiogenesis during SK and since Src kinases are involved in signaling by many of them, the use of Src kinase inhibition represents a promising way of limiting the severity of SK lesions the most common cause of infectious blindness in the Western world.     

Sphingomyelin Patches on Pancreatic Beta-cells Are Indicative of Insulin Secretory Capacity

The establishment and validation of specific markers on the surfaces of pancreatic beta-cells would have a significant impact on the development of agents that specifically target these cells for imaging and/or image-guided therapy in diabetes patient samples. We have recently described unique, cholesterol-stabilized sphingomyelin (SM) patches on the surfaces of beta-cells using the IC2 antibody. To further investigate the utility of SM patches as a unique beta-cell biomarker, we embarked on the current study to correlate the expression of this antigen with the insulin secretory capacity of beta-cells in tissue samples from patients and animals with type 1 and type 2 diabetes and compared this with samples from normal subjects. We found that the locations of SM patches were consistent with the insulin status of islets in all tissues studied. Using immunohistochemistry and staining with an IC2 antibody, we demonstrated a direct correlation between the reduced expression of SM patches and insulin production in diabetic individuals, indicating that the former could potentially serve as a functional biomarker of beta-cells. We believe that our results have significant implications for the further development of ligands with SM specificity for the non-invasive functional assessment of beta-cells and/or for targeted therapeutic delivery in diabetic patients.